QP 

601 


MONOGRAPHS   ON   BIOCHEMISTRY 

EDITED    BY 

R.    H.   ADERS   PLIMMER,    D.Sc. 

AND 

F.    G.    HOPKINS,    M.A.,    M.B.,    D.Sc.,   F.R.S. 


MONOGRAPHS  ON  BIOCHEMISTRY. 

THE  DEVELOPMENT  AND  PRESENT  POSI- 
TION OF  BIOLOGICAL  CHEMISTRY.  By 
F.  GOWLAND  HOPKINS,  M.A.,  M.B.,  D.Sc.,  F.R.S. 

THE  NATURE  OF  ENZYME  -  ACTION.  By 
W.  M.  BAYLISS,  D.Sc.,  F.R.S. 

THE  CHEMICAL  CONSTITUTION  OF  THE 
PROTEINS.  By  R.  H.  ADERS  PLIMMER,  D.Sc. 

THE  GENERAL  CHARACTERS  OF  THE  PRO- 
TEINS. By  S.  B.  SCHRYVER,  D.Sc.,  Ph.D. 

THE  VEGETABLE  PROTEINS.      By  THOMAS  B. 

OSBORNE. 

THE  CARBOHYDRATES:  THE  POLYSACCHA- 
RIDES.  By  ARTHUR  R.  LING,  F.I.C. 

THE  CARBOHYDRATES:  THE  GLUCOSIDES. 
By  E.  FRANKLAND  ARMSTRONG,  D.Sc.,  Ph.D. 

THE  FATS.     By  J.  B.  LEATHES,  D.Sc. 


THE     NATURE 


OF 


ENZYME      ACTION 


BY 


W.  M.  BAYLISS,  D.Sc.,  F.R.S. 
?f 

ASSISTANT  PROFESSOR    OF  PHYSIOLOGY,    UNIVERSITY   COLLEGE,  LONDON 


LONGMANS,      GREEN,      AND      CO. 

39  PATERNOSTER  ROW,  LONDON 

NEW  YORK,  BOMBAY,  AND  CALCUTTA 

1908 


,-. 


OF  THE 

UNIVERSITY 

pi 


GENERAL   PREFACE. 

THE  subject  of  Physiological  Chemistry,  or  Biochemistry,  is 
enlarging  its  borders  to  such  an  extent  at  the  present  time, 
that  no  single  text-book  upon  the  subject,  without  being 
cumbrous,  can  adequately  deal  with  it  as  a  whole,  so  as  to 
give  both  a  general  and  a  detailed  account  of  its  present 
position.  It  is,  moreover,  difficult,  in  the  case  of  the  larger 
text-books,  to  keep  abreast  of  so  rapidly  growing  a  science 
by  means  of  new  editions,  and  such  volumes  are  therefore 
issued  when  much  of  their  contents  has  become  obsolete. 

For  this  reason,  an  attempt  is  being  made  to  place  this 
branch  of  science  in  a  more  accessible  position  by  issuing 
a  series  of  monographs  upon  the  various  chapters  of  the 
subject,  each  independent  of  and  yet  dependent  upon  the 
others,  so  that  from  time  to  time,  as  new  material  and 
the  demand  therefor  necessitate,  a  new  edition  of  each  mono- 
graph can  be  issued  without  re-issuing  the  whole  series.  In 
this  way,  both  the  expenses  of  publication  and  the  expense 
to  the  purchaser  will  be  diminished,  and  by  a  moderate 
outlay  it  will  be  possible  to  obtain  a  full  account  of  any 
particular  subject  as  nearly  current  as  possible.  *' 

The  editors  of  these  monographs  have  kept  two  objects 
in  view  :  firstly,  that  each  author  should  be  himself  working 
at  the  subject  with  which  he  deals ;  and,  secondly,  that  a 
Bibliography,  as  complete  as  possible,  should  be  included, 


193628 


vi  GENERAL  PREFACE 

in  order  to  avoid  cross  references,  which  are  apt  to  be 
wrongly  cited,  and  in  order  that  each  monograph  may  yield 
full  and  independent  information  of  the  work  which  has  been 
done  upon  the  subject. 

It  has  been  decided  as  a  general  scheme  that  the  volumes 
first  issued  shall  deal  with  the  pure  chemistry  of  physiological 
products  and  with  certain  general  aspects  of  the  subject. 
Subsequent  monographs  will  be  devoted  to  such  questions 
as  the  chemistry  of  special  tissues  and  particular  aspects  of 
metabolism.  So  the  series,  if  continued,  will  proceed  from 
physiological  chemistry  to  what  may  be  now  more  properly 
termed  chemical  physiology.  This  will  depend  upon  the 
success  which  the  first  series  achieves,  and  upon  the  divisions 
of  the  subject  which  may  be  of  interest  at  the  time. 

R.    H.    A.    P. 
F.   G.    H. 


PREFACE. 

THIS  short  monograph  is  based  upon  lectures  given  at 
various  times  in  University  College,  London,  and  this  fact 
may  serve  as  the  excuse  for  what  some  may  regard  as  too 
definite  a  position  with  respect  to  certain  disputed  questions. 
It  is,  I  venture  to  think,  preferable  to  take  a  definite  point  of 
view,  rather  than  to  leave  the  study  of  the  subject  in  a 
state  of  chaos.  At  the  same  time,  one  must  always  be 
prepared  to  correct  one's  views  when  evidence  is  brought 
against  them.  I  have  endeavoured  not  to  slur  over  diffi- 
culties, where  such  really  exist,  as  also  to  indicate  the 
existence  of  points  of  view  contrary  to  my  own.  It  is 
quite  possible,  however,  that,  by  inadvertence  or  ignorance, 
important  facts  may  have  been  omitted. 

Certain  subjects  of  considerable  interest  in  connection 
with  the  theory  of  the  action  of  enzymes,  such  as  the  pro- 
perties of  colloids  and  the  laws  of  adsorption,  which  were 
dealt  with  in  some  detail  in  the  lectures,  are  here  only 
treated  in  their  immediate  bearing  on  the  facts  under  dis- 
cussion. 

No  attempt  has  been  made  to  put  forward  a  complete 
bibliography  in  the  list  of  literature  at  the  end.  This  contains 
only  those  writings  which  have  a  more  or  less  direct  bearing 
on  the  subject  and  which  are  referred  to  in  the  text.  In  this 
list  I  have  added,  after  each  reference,  the  page  of  the  text 

b 


viii  PREFACE 

in  which  it  is  made  use  of,  so  that  the  insertion  of  names  of 
authors  in  the  general  index  is  rendered  unnecessary. 

The  detailed  description  of  the  properties  of  the  large 
number  of  specific  enzymes  already  known  finds  no  place 
in  this  monograph,  which  confines  itself  to  general  pro- 
perties more  or  less  common  to  all  enzymes. 

W.    M.    B. 


CONTENTS. 

CHAPTER  AGE 

I.  CATALYSIS  IN  GENERAL  -  i 

II.  ENZYMES  AS  CATALYSTS  -  6 

III.  CHEMICAL  AND  PHYSICAL  PROPERTIES  OF  ENZYMES  -       13 

IV.  GENERAL  METHODS  OF  PREPARATION  AND  OF  INVESTIGATION  -       21 
V.  REVERSIBILITY  OF  ENZYME-ACTION  -  -       28 

VI.  THE  VELOCITY  OF   REACTION   AND   THE   VARIOUS   CONDITIONS 

AFFECTING  IT  36 

VII.  THE    NATURE   OF   THE   COMBINATION   BETWEEN    ENZYME  AND 

SUBSTRATE      -  r       59 

VIII.  CO-ENZYMES  AND  ANTI-ENZYMES     -  65 

IX.  ZYMOGENS       -  69 

X.  OXIDATION-PROCESSES  AND  CERTAIN  COMPLEX  SYSTEMS    -  -       71 

GENERAL  CONCLUSIONS  -  -       75 

SUPPLEMENTARY  NOTES  -  76 

LIST  OF  LITERATURE  REFERRED  TO  -       78 

INDEX   -                           -..__.  -       87 


CHAPTER  I. 

CATALYSIS  IN  GENERAL. 

ONE  of  the  most  striking  characteristics  of  the  chemical  changes  taking 
place  in  living  organisms  is  the  ease  with  which  bodies  of  a  highly  stable 
nature  are  split  up.  Glucose,  for  example,  is  oxidised  to  carbon  dioxide 
and  water,  egg-white  is  hydrolysed  to  amino-acids.  Under  ordinary 
laboratory  conditions,  powerful  reagents,  such  as  chromic  acid  and 
boiling  hydrochloric  acid,  are  necessary  to  effect  these  decompositions.1 
This  fact,  which  is,  of  course,  familiar  to  all  workers  in  bio-chemistry, 
was,  at  an  early  date  in  the  history  of  the  science,  especially  called 
attention  to  by  Schonbein  (142,  p.  344). 

Phenomena  of  a  similar  kind  are,  however,  known  to  chemists  to 
take  place  in  the  laboratory,  and  such  reactions  are  known  as  "  cata- 
lytic "  reactions.  They  are  increasing  in  number  and  importance  every 
day.  Oxygen  and  hydrogen,  for  instance,  at  ordinary  temperatures 
combine  so  slowly  that  the  production  of  water  cannot  be  detected,  the 
application  of  a  flame  or  electric  spark  being  requisite.  But  the  pre- 
sence of  a  minute  quantity  of  finely  divided  platinum  is  sufficient  to 
cause  combination  to  take  place  at  room  temperature.  Again,  the  oxi- 
dations effected  by  hydrogen  peroxide  proceed  in  many  cases  at  a  very 
slow  rate  by  themselves,  but  can  be  enormously  accelerated  by  traces  of 
iron  or  manganese,  as  m  the  well-known  method  of  Fenton  (62). 
Another  case  of  interest  in  connection  with  enzyme-action  is  the 
hydrolysis  of  cane-sugar  by  acids  (ionic  hydrogen). 

The  most  profitable  way  of  studying  the  problem  before  us  is  to 
consider  first  of  all  the  essential  characters  of  catalysis,  as  manifested 
by  reactions  where  the  bodies  concerned  are  of  known  chemical  com- 
position. 

For  this  purpose  we  may  conveniently  divide  reactions  into  two 
classes. 

I.  There  are  a  large  number  of  reactions  which  are  practically  in- 
stantaneous, those  between  ions  forming  the  chief  part  of  this  class. 
When  a  chloride  is  added  to  a  solution  of  silver  nitrate,  a  precipitate  of 
silver  chloride  falls  at  once.     Or,  when  a  strong  acid  is  neutralised  by 
a  strong  base,  the  union  takes  place  at  once,  as  we  know  by  the  regular 
titration  methods. 

II.  On  the  other  hand,  there  are  reactions,  like  the  saponification  of 
esters  by  caustic  alkali,  which  take  a  measurable  time  to  arrive  at  their 
final  state. 

Now,  a  "  catalyst "  is  a  body  which  alters  the  rate  of  reactions  of 
this  latter  class  (see  Ostwald  [i  27,  i.,  p.  5 1 5]).  The  change  may  be  either 

1  Note  A  at  end  of  book, 
I 


2  THE  NATURE  OF  ENZYME-ACTION 

in  the  direction  of  acceleration  or  of  retardation,  and  the  reaction  may 
be  one  that,  by  itself,  either  proceeds  rapidly  or  so  slowly  that  it  re- 
quires special  proof  to  show  that  it  is  taking  place  at  all.  It  is  especi- 
ally to  the  acceleration  of  this  latter  kind  of  reaction  that  the  name  of 
catalysis  is  usually  given,  although,  in  theory,  any  change  of  the  rate  of 
any  reaction  by  the  addition  of  a  foreign  substance  comes  under  the 
same  category. 

As  examples  of  catalysed  reactions  may  be  mentioned  :  the  inver- 
sion (hydrolysis)  of  cane-sugar  by  acid  (hydrion),  the  numerous  com- 
binations effected  by  the  catalytic  agency  of  platinum  in  particulate 
condition,  and  oxidations  by  hydrogen  peroxide  accelerated  by  ferrous 
or  manganous  salts.  As  a  case  of  slowing  of  a  reaction  by  a  foreign 
body  I  may  refer  to  the  stopping  of  the  slow  oxidation  of  phosphorus 
in  air  by  a  trace  of  ether  vapour ;  this  kind  of  action  is  called  "  negative 
catalysis  ". 

There  are  certain  phenomena  which,  at  first  sight,  might  be  con- 
fused with  those  of  catalysis,  but  which  must  be  carefully  distinguished 
from  them.  A  mechanical  model  will  serve  to  make  this  clear.  If  a 
brass  weight  of,  say,  500  grammes  be  placed  at  the  top  of  an  inclined 
plane  of  polished  plate-glass,  it  will  be  possible  to  find  a  slope  of  the 
plane  such  that  the  weight  will  slowly  slide  down.  This  represents  any 
reaction  taking  time  to  complete.  If  now  the  bottom  of  the  weight  be 
oiled  (oil  =  catalyst)  the  rate  of  its  fall  will  be  greatly  increased.  We 
see  that,  in  either  case,  the  weight,  if  placed  at  the  top  of  the  plane, 
does  not  remain  there,  but,  sooner  or  later,  reaches  the  bottom.  It  may, 
however,  be  kept  at  the  top  by  some  kind  of  catch  or  trigger  arrange- 
ment, in  which  case  it  will  remain  there  indefinitely  until  the  catch  is 
released.  The  amount  of  energy  lost  by  the  weight  in  its  fall,  being 
the  product  of  its  weight  and  the  vertical  height  from  which  it  has 
fallen,  is  in  no  way  affected  by  the  work  required  to  remove  the  obstacle 
preventing  its  fall,  nor  is  the  rate  at  which  it  falls  when  set  free.  A 
typical  instance  of  such  a  "  trigger "  action  is  that  of  supersaturated 
solutions,  which  remain  for  any  length  of  time  unchanged  unless  infected 
with  a  crystal.  It  has,  moreover,  been  shown  by  B.  Moore  (119)  that 
the  rate  at  which  the  solidification  of  supercooled  glacial  acetic  acid 
moves  along  a  tube  is  independent  of  the  quantity  of  crystals  placed  at 
one  end  to  start  the  process.  Not  so  with  true  catalytic  action  ;  al- 
though the  work  done  by  our  sliding  weight  is  in  no  way  affected  by 
the  amount  of  catalyst  (oil)  used,  the  rate  of  the  fall  is  within  limits 
directly  proportional  to  it,  and  this  is  a  property  of  catalysts  in  general. 

It  cannot  be  expected  that  a  rough  model  of  this  kind  would  show 
all  of  the  characteristics  of  catalytic  phenomena,  but  there  are  two  in- 
structive points  shown  by  it  in  addition  to  those  already  spoken  of. 
The  first  is  the  disappearance  of  the  catalyst  by  sticking  to  the  glass  as 
the  weight  slides  down.  An  analogous  phenomenon  is  often  met  with 
in  catalytic  processes,  as  will  be  seen  later.  The  second  point  is  one  of 
importance  with  regard  to  certain  enzyme-actions ;  it  consists  in  the 
fact  that,  although  the  presence  of  the  catalyst  neither  adds  to  nor  sub- 
tracts from  the  total  energy  change  in  the  reaction,  the  form  of  this 
energy  may  be  altered.  When  the  weight  falls  slowly  by  itself,  nearly 
the  whole  of  the  energy  appears  as  heat  due  to  friction  along  the  glass 
plane,  so  that  the  weight  arrives  at  the  bottom  with  very  little  kinetic 


CATALYSIS  IN  GENERAL  3 

energy ;  on  the  contrary,  when  oiled,  nearly  the  whole  of  the  energy  is 
present  in  the  weight  at  the  end  of  its  fall  as  kinetic  energy,  very  little 
friction  having  been  met  with  in  its  descent. 

From  what  has  been  said  it  follows  that  a  catalyst  is  merely  cap- 
able of  changing  the  rate  of  a  reaction  already  in  progress.  In  opposi- 
tion to  this  it  may  reasonably  be  said  that  a  reaction  does  sometimes 
seem  to  be  initiated.  Such  a  case  is  that  of  a  mixture  of  oxygen  and 
hydrogen  gases  caused  to  combine  by  spongy  platinum.  Now  there 
are  reasons  for  the  belief  that  an  extremely  slow  combination  is  taking 
place  at  ordinary  temperatures  without  catalysis.  One  thing  to  be  con- 
sidered in  reference  to  this  belief  is  the  enormous  acceleration  of  chemical 
reactions  by  rise  of  temperature,  the  majority  being  about  doubled  by  a 
rise  of  10°  C.  In  this  way  a  reaction  having  a  velocity  of  I  at  o°  would- 
reach  one  of  2  at  10°,  4  at  20°  and  I  x  210  =  1,024  at  100°.  At  the 
temperature  of  500°  there  is  appreciable  formation  of  water  in  the  case 
in  point,  and  Bodenstein  (30,  pp.  694  and  68 1)  has  shown  that,  if  the 
velocity  at  689°  be  represented  by  163,  that  at  482°  has  already  sunk  to 
0*28  ;  so  that  at  room  temperature  the  velocity  would  be  quite  incapable 
of  detection  by  chemical  means,  since  centuries  would  be  needed  to  pro- 
duce a  fraction  of  a  milligramme  of  water.  Grove's  gas  battery  also  proves 
that  the  two  gases  are  not  in  equilibrium  at  ordinary  temperatures,  since 
electrical  energy  is  obtained  by  their  slow  combination.  Other  cases 
will  be  better  considered  when  discussing  catalysis  by  enzymes. 

Several  other  properties  of  catalysts  must  be  referred  to.  They  do 
not  appear  chemically  combined  with  the  final  products  of  the  reaction, 
which  are,  as  a  rule,  the  same  as  those  of  the  non-catalysed  reaction. 
They  are  found  at  the  end  unchanged,  except  in  those  cases  in  which 
they  are  destroyed  by  subsidiary  reactions,  such  as  the  nitric  acid  in  the 
old  chamber  process  of  sulphuric  acid  manufacture. 

Since  the  addition  of  a  catalyst  only  serves  to  hasten  a  result  which 
would  ultimately  be  arrived  at  by  itself,  it  follows  that  the  effect  of  a 
small  quantity  of  catalyst  is,  in  the  end,  the  same  as  that  of  a  larger 
quantity,  if  sufficient  time  be  allowed.  This  is  often  in  practice  a  use- 
ful criterion  in  deciding  whether  a  result  produced  by  the  addition  of  a 
body  is  due  to  a  chemical  reaction  following  the  laws  of  combining  pro- 
portion, or  to  a  catalytic  process.  In  the  first  case  the  amount  of  the 
new  products  formed  will  be  in  exact  ratio  to  the  quantity  of  the  body 
added,  in  the  second  case  they  will  be  independent  of  this  factor;  the 
only  difference  between  the  reactions  when  large  or  small  amounts  of 
the  foreign  body  are  added,  if  it  acts  as  a  catalytic  agent,  is  the  rate  at 
which  the  new  products  appear.  Of  course,  this  last  statement  is  true 
only  when  the  catalyst  is  not  paralysed  or  destroyed  before  it  has  com- 
pleted its  work.  Cases  are  known,  in  fact,  where  such  phenomena 
happen,  so  that  the  final  result  depends  on  the  amount  of  catalyst 
added  ;  such  cases  are  not  very  infrequent  amongst  enzyme-actions  and 
will  be  considered  later. 

Although  the  degree  of  acceleration  of  a  reaction  is  proportional  to 
the  concentration  of  catalyst  present,  it  is  astonishing  how  minute  a 
quantity  is  capable  of  perceptible  activity.  For  example,  according  to 
Bredig  and  v.  Berneck  (35,  p.  276),  colloidal  platinum  is  able  to  act  upon 
1,000,000  times  its  weight  of  hydrogen  peroxide;  again,  it  was  found 
by  Erode  (36,  p.  289)  that,  in  the  reaction  between  hydrogen  peroxide 

I  * 


4  THE  NATURE  OF  ENZYME-ACTION 

and  hydriodic  acid,  the  catalytic  action  of  I  gramme-molecule  of  molybdic 
acid  in  31,000,000  litres  could  still  be  detected. 

The  question  naturally  arises,  when  the  reaction  catalysed  is  a  rever- 
sible, or  balanced,  one,  has  the  fact  that  the  equilibrium  position  is  ar- 
rived at  under  the  accelerating  influence  of  a  catalyst  any  effect  on  the 
actual  position  of  this  equilibrium  ?  It  was  shown  by  Berthelot  and 
Pean  de  Saint-Gilles  (24,  p.  418)  that,  if  i  mol.  (  =  gramme-molecule)  of 
ethyl  alcohol  be  mixed  with  I  mol.  of  acetic  acid,  the  reaction  proceeds 
until  the  mixture  has  the  composition  : — 

|  mol.  alcohol  +  £  mol.  acid  +  f  mol.  ester  +  f  mol.  water. 
The  same  end-position  is  also  reached  when  I  mol.  of  ethyl  acetate  is 
mixed  with  I  mol.  of  water.  So  that,  in  any  mixture  of  these  four 
bodies,  two  opposite  reactions  are  proceeding  at  unequal  rates  until  a 
certain  relative  concentration  of  the  constituents  results,  at  which  point 
the  two  reactions  have  an  equal  velocity. 

Now  suppose  that  a  catalyst,  e.g.,  HC1,  is  added  to  such  a  system, 
it  is  plain  that,  unless  both  the  opposite  reactions  were  equally  accelerated, 
the  equilibrium-position  would  be  changed.  This  can  only  be  done  by 
adding  or  taking  away  energy,  since  a  change  in  the  position  of  equi- 
librium implies  a  change  in  the  osmotic  pressure  of  the  solution.  If, 
then,  our  definition  of  a  catalyst  be  correct,  viz.,  that  it  neither  adds  nor 
subtracts  energy,  it  follows  that  it  must  accelerate  both  the  hydrolytic 
and  the  synthetic  reactions.  This  has  been  shown  experimentally  by 
Knoblauch  (101,  p.  269)  for  the  cases  of  formation  and  decomposition 
of  esters  by  hydrochloric  acid.  As  regards  the  non-alteration  of  the 
position  of  equilibrium,  the  proof  has  been  given  by  Koelichen  (102,  pp. 
136  and  149),  in  the  case  of  the  reversible  polymerisation  of  acetone  by 
the  catalytic  action  of  bases,  that  the  equilibrium-constant  is,  in  dilute 
solution,  independent  of  the  concentration  or  nature  of  the  catalysing  base. 
Similarly,  it  has  been  shown  by  Turbaba  (157)  that  the  equilibrium  be- 
tween aldehyde  and  paraldehyde  is  independent  of  the  nature  or  amount 
of  the  catalyst,  whether  this  be  sulphur  trioxide,  zinc  sulphate,  hydro- 
chloric, oxalic  or  phosphoric  acids. 

The  state  of  affairs  described  above  does  not  necessarily  hold  when 
the  catalyst  is  altered  in  any  way  by  the  reaction ;  such  alteration  may 
be  physical  or  chemical.  The  former  case  is  of  special  importance  as 
concerning  enzymes,  as  will  appear  later.  It  has  also  been  shown  by 
Abel  (3  and  185)  that,  when  the  reaction  proceeds  with  the  formation 
of  an  intermediate  combination  between  the  catalyst  and  the  body  which 
is  attacked,  the  law  that  catalysts  must  raise,  in  quantitatively  equal  pro- 
portional amount,  the  two  opposite  factors  of  a  chemical  reaction,  does 
not  necessarily  hold.  In  other  words,  if  the  catalyst  is  in  a  different 
chemical  or  physical  state  at  the  end  from  what  it  was  at  the  beginning, 
it  has  given  up  or  received  energy,  so  that  a  change  of  position  of  equi- 
librium is  quite  conceivable. 

It  seems  probable,  indeed,  that  the  explanation  of  a  large  number  of 
catalytic  reactions  lies  in  this  formation  of  intermediate  compounds.  At 
the  same  time,  as  Ostwald  (see  Erode  [36,  p.  305])  has  pointed  out,  in 
order  that  this  shall  be  a  satisfactory  theory  it  must  be  shown,  in  any 
given  case,  that  the  sum  of  all  the  reactions  when  intermediate  compounds 
are  formed  is  more  rapid  than  the  uncatalysed  reaction  itself.  Erode 
(36,  p.  281,  etc.)  has  given  the  direct  proof  of  one  case  of  such  a  reaction. 


CATALYSIS  IN  GENERAL  5 

Hydriodic  acid  is  decomposed  by  hydrogen  peroxide  at  a  measurable 
rate,  which  is  enormously  increased  by  molybdic  acid  acting  as  a  cata- 
lyst. During  the  course  of  the  reaction  a  series  of  permolybdic  acids 
can  be  shown,  by  chemical  means,  to  be  formed  and  the  velocity  of  their 
production  determined.  These  acids  decompose  hydriodic  acid  with 
great  rapidity,  so  that  the  sum  of  the  two  reactions,  formation  of  per- 
molybdic acids  by  action  of  hydrogen  peroxide  on  molybdic  acid  and 
decomposition  of  hydriodic  acid  by  these,  take  place  at  a  considerably 
greater  rate  than  that  at  which  hydrogen  peroxide  by  itself  can  effect 
the  change. 

The  theory  of  intermediate  compounds  does  not  explain  all  cases. 
For  example,  Tafel  (151)  has  shown  that  the  catalytic  action  of  hydro- 
chloric acid  in  the  formation  of  esters  from  methyl  alcohol  and  acids 
cannot  be  accounted  for  by  the  intermediate  production  of  methyl 
chloride  (CH3C1),  as  had  been  suggested.  In  the  first  place,  this  body 
is  not  formed  with  any  rapidity  under  the  conditions  of  the  reaction,  and, 
in  the  second  place,  when  added,  it  is  found  unable  to  replace  hydro- 
chloric acid  as  a  catalyst. 

It  would  be  unfruitful,  in  the  present  state  of  knowledge,  to  discuss 
in  further  detail  the  various  hypotheses  put  forward  to  explain  catalysis. 
One  of  these,  however,  is  of  importance  in  connection  with  colloidal 
catalysts,  such  as  enzymes  are,  namely,  surface-condensation  of  the  re- 
acting bodies,  in  which  case  the  accelerated  rate  of  change  is,  in  all  prob- 
ability, due  to  increase  of  concentration.  A  similar  hypothesis  was 
suggested  by  Faraday  to  explain  the  action  of  platinum  on  mixtures  of 
oxygen  and  hydrogen  gases.1 

Attention  must  be  called  to  one  more  point  before  passing  on  to  the 
consideration  of  the  special  class  of  catalysts  known  as  "  enzymes  "  ;  in 
view  of  certain  theories  as  to  the  nature  of  enzymes,  it  is  important  to 
notice  that  all  the  catalysts  mentioned  in  this  chapter  are  definite 
chemical  individuals,  of  known  composition  and  properties.  As  yet 
this  statement  cannot  be  made  of  any  enzyme.  We  are  not,  however, 
warranted  in  denying  definite  chemical  constitution  to  them,  until  it  has 
been  shown  that  bodies  of  known  constitution  may  at  one  time  possess 
the  properties  of  enzymes  and  at  another  time,  without  any  change  in 
their  chemical  nature,  be  devoid  of  such  properties. 

For  a  more  detailed  account  of  the  general  phenomena  of  catalysis, 
Chapters  X.  and  XL  of  Mellor's  Chemical  Statics  and  Dynamics  should 
be  consulted. 

1  See  Mellor's  Chemical  Statics  and  Dynamics,  p.  258,  etc. 


CHAPTER  II. 

ENZYMES  AS  CATALYSTS. 

AT  an  early  date  in  the  history  of  physiology  bodies  having  properties 
similar  to  those  of  the  inorganic  catalysts  were  prepared  from  the  tissues 
of  living  organisms.  In  1830  Dubrunfaut  prepared  an  extract  of  malt 
which  converted  starch  into  sugar  (49),  just  as  strong  acids  were  known 
to  do  (Kirchhoff  [97]).  Three  years  later,  Payen  and  Persoz  precipi- 
tated by  alcohol  from  such  extracts  a  substance  which  could  be  dried 
and  preserved,  and  which  had  a  very  powerful  action  on  starch  (130). 
This  they  called  "  diastase  ". 

As  more  bodies  of  similar  properties  became  known,  they  were 
called  "  ferments,"  on  account  of  the  resemblance  of  their  activities  to 
those  of  alcoholic  fermentation. 

When  Pasteur  had  shown  that  this  process  was  due  to  the  presence 
of  a  living  organism,  diastase  and  bodies  like  it  were  distinguished  as 
" soluble  or  unorganised  ferments"  in  contradistinction  to  living  or- 
ganisms, like  yeast,  which  went  by  the  name  of  "organised  ferments". 

Although  many  physiologists,  as  Traube  for  example,  were  of 
opinion  that  organised  ferments  owed  their  properties  to  the  presence 
within  them  of  soluble  ferments,  the  double  use  of  the  word  "  ferment " 
gave  rise  to  some  confusion,  and  induced  Kiihne  (105,  p.  293)  to  suggest 
a  new  name  in  an  interesting  passage,  of  which  the  translation  is  :— 

"  The  latter  designations  (z>.,  formed  and  unformed  ferments)  have 
not  gained  general  acceptance,  in  that  on  the  one  hand  it  was  objected 
that  chemical  bodies,  like  ptyalin,  pepsin,  etc.,  could  not  be  called  fer- 
ments, since  the  name  was  already  given  to  yeast-cells  and  other 
organisms  (Briicke) ;  while  on  the  other  hand  it  was  said  that  yeast-cells 
could  not  be  called  ferment,  because  then  all  organisms,  including  man, 
would  have  to  be  so  designated  (Hoppe-Seyler).  Without  stopping  to 
inquire  further  why  the  name  excited  so  much  opposition,  I  have  taken 
the  opportunity  to  suggest  a  new  one,  and  I  give  the  name  enzymes  to 
some  of  the  better  known  substances,  called  by  many  *  unformed  fer- 
ments '.  This  is  not  intended  to  imply  any  particular  hypothesis,  but  it 
merely  states  that  eV  ZV/JLV)  (in  yeast)  something  occurs  that  exerts  this 
or  that  activity,  which  is  considered  to  belong  to  the  class  called  fer- 
mentative. The  name  is  not,  however,  intended  to  be  limited  to  the 
invertin  of  yeast,  but  it  is  intended  to  imply  that  more  complex  organ- 
isms, from  which  the  enzymes,  pepsin,  trypsin,  etc.,  can  be  obtained, 
are  not  so  fundamentally  different  from  the  unicellular  organisms  as 
some  people  would  have  us  believe." 

In  this  monograph  the  name  "enzyme"  will  therefore  be  invariably 
used. 


ENZYMES  AS  CATALYSTS  7 

We  may,  for  the  present  then,  define  enzymes  as  the  catalysts 
produced  by  living  organisms.  This  statement  is  not  to  be  taken  as 
in  any  way  prejudging  the  possibility  of  their  ultimate  production  in 
the  laboratory  ;  when  a  body  of  the  properties  of  trypsin  is  synthesised 
it  will  have  every  right  to  the  same  name.  In  the  actual  investigation 
of  enzymes  their  source  is,  of  course,  immaterial. 

In  the  above  definition,  as  will  be  noted,  it  is  assumed  that  enzymes 
behave  as  catalysts,  so  that  it  is  incumbent  upon  us  at  the  outset  to 
consider  how  far  the  statement  is  justified. 

Berzelius  (27),  in  his  Lehrbuch  der  Chemie^  had  already  called  atten- 
tion to  the  similarity  of  enzymes  and  catalysts  in  the  following  words : 
"We  had  the  experience  of  finding  that  the  change  of  sugar  into 
carbon  dioxide  and  alcohol,  as  it  takes  place  in  fermentation  under  the 
influence  of  an  insoluble  body,  which  we  know  by  the  name  of  ferment, 
could  not  be  explained  by  an  action  similar  to  double  decomposition 
between  the  sugar  and  ferment.  But,  when  compared  with  known 
phenomena  in  the  inorganic  world,  it  resembled  nothing  else  so  much 
as  the  decomposition  of  hydrogen  peroxide  under  the  influence  of 
platinum,  silver  or  fibrin ;  it  was  therefore  quite  natural  to  assume  an 
analogous  action  in  the  case  of  the  ferment."  And  again  :  "  We  have 
reasons,  well  founded  on  fact,  to  make  the  assertion,  that  in  living 
plants  and  animals  there  take  place  thousands  of  catalytic  processes 
between  tissues  and  fluids". 

Views  of  a  similar  kind  have  been  expressed  by  physiologists,  Carl 
Ludwig,  Traube,  Bunge  and  many  others. 

Before  proceeding  further  with  the  question  before  us,  it  is  advisable 
to  briefly  refer  to  the  terminology  of  enzyme-action  in  general.  A 
name  is  frequently  needed  for  the  substances  on  which  enzymes  exert 
their  activity.  Unfortunately,  no  really  satisfactory  name  has  yet  been 
suggested.  "  Hydrolyte  "  would  serve  where  the  action  is  one  of  hydro- 
lysis, but  it  would  exclude  any  other  action  such  as  oxidation  or  intra- 
molecular splitting,  as  well  as  the  synthetic  actions,  such  as  the 
formation  of  a  disaccharide  by  the  action  of  maltase  on  glucose.  On 
the  whole,  "  substrate,"  already  used  by  many  writers,  seems  to  answer 
the  purpose  best. 

As  to  the  names  of  the  enzymes  themselves,  it  was  suggested  by 
Duclaux  that  the  termination  "  ase "  should  be  taken  as  denoting  an 
enzyme  and  that  this  termination  should  be  added  to  the  name  of  the 
substrate,  e.g.y  lactase  is  the  enzyme  accelerating  the  hydrolysis  of 
lactose.  It  would  be  inconvenient  to  displace  old-established  names,  such 
as  "  pepsin  "  and  "  trypsin,"  but,  as  far  as  possible,  the  recommendation 
of  Duclaux  should  be  acted  upon. 

It  has  been  the  custom  to  speak  of  an  enzyme  which  attacks,  say, 
starch  or  protein,  as  "  amylolytic  "  or  "  proteolytic  "  respectively  ;  but,  as 
Professor  Armstrong  has  pointed  out,  these  names  are  incorrectly  formed. 
"  Amylolytic,"  in  analogy  with  "electrolytic,"  should  mean  a  decomposi- 
tion by  means  of  starch.  To  avoid  this  misuse  of  words,  Professor  Arm- 
strong (7)  advocates  the  use  of  the  termination  "clastic"  instead  of 
"  lytic  "  in  speaking  of  the  action  of  an  enzyme  on  its  appropriate  sub- 
strate. 

Apart  from  theory,  it  is  useful  to  know  what  kind  of  properties  to 
look  for  in  a  substance  suspected  to  be  an  enzyme.  An  unknown  body, 


8  THE  NATURE  OF  ENZYME-ACTION 

if  an  enzyme,  may  be  expected  to  manifest  the  general  characters  of 
catalysts  to  a  greater  or  less  degree. 

•^-  Now  we  have  seen  that  there  are  practically  only  two  properties 
common  to  all  catalysts,  viz.,  that  of  not  initiating  a  new  reaction,  but 
merely  changing  the  rate  of  one  already  in  progress,  and  that  of  not 
appearing  in  the  final  products  of  the  reaction.  Certain  other  properties 
either  follow  from  these,  or,  although  possessed  by  the  majority  of  cata- 
lysts, do  not  seem  to  be  essential.  We  will  now  proceed  to  examine, 
in  order,  these  various  properties  as  manifested  by  enzymes. 

It  is  obviously  not  an  easy  matter  to  give  a  satisfactory  answer  to  the 
question  whether  enzymes  start  a  new  reaction  or  accelerate  one  already 
in  progress.  The  state  of  affairs  is,  in  fact,  similar  to  that  of  a  mixture 
of  oxygen  and  hydrogen  gases  catalysed  by  platinum,  in  which  we  found 
evidence  that  the  combination  does  take  place  at  room  temperatures, 
although  at  an  inappreciable  rate.  The  greater  number  of  enzymes  have 
a  hydrolytic  action,  and  their  activities  are,  as  a  rule,  manifested  in  the 
presence  of  an  excess  of  water.  Now  we  know  that  water  contains 
hydrogen-  and  hydroxyl-ions,  which  have  a  hydrolytic  action  and, 
although  their  concentration  is  very  small  at  o°,  it  increases  rapidly  as 
the  temperature  rises.  The  following  numbers  will  serve  to  show 
this:— 

Gramme-ions  of  H*  per  litre  of  water  at : — 
o°  0*35  x   io~7 

18°       .        .         .     0-80  x   io~7 
50°       ...     2-48  x   io-7(Kohlrausch). 

In  fact,  water  at  100°  is  capable  of  hydrolysing  cane-sugar  to  glucose 
and  fructose  at  a  measurable  rate.     It  is,  therefore,  no  unjustifiable  as- 
sumption that  the  process  takes  place  at  room  temperature,  although 
slowly.     Changes  at  this  temperature  have,  moreover,  been  described. 
Starch  solutions  were  found  by  Aggazzotti  (4)  to  undergo  a  spontane- 
ous change  in  the  direction  of  dextrin  and  sugar.     Brailsford  Robertson 
I  (I37>  P-  344))  also,  noticed  that  solutions  of  ammonium  caseinogenate 
j   slowly  increased  in  electrical  conductivity  when  left  to  themselves,  a 
f    change  similar  to  that  which  occurs  when   they  are  acted  upon   by 
trypsin. 

Certain  difficulties  must  not  be  overlooked.  Enzymes,  in  many 
jucases,  do  not  carry  the  hydrolytic  process  as  far  as  acids  do.  The 
amylase  of  malt  converts  starch  into  maltose  and  apparently  no  further, 
whereas  acids  convert  it  into  glucose ;  frrypgin  ^rf.q  pn  prntQJr|«;  Wvin|r 
unattacked  a  complex  polypeptide^  which  can  he  further  split  info 
amino-acids  by  acids  or  by  the  enzyme,  ercpsin.  Other  cases  might 
6"e~ mentioned,  but  these  will  suffice  to  illustrate  the  point.  With  re- 
gard to  this  objection,  there  are  one  or  two  considerations  to  be  borne 
in  mind  which  tend  to  remove  its  serious  nature  in  some  degree.  In 
the  first  place,  it  is  not  impossible  that  all  these  reactions  would  continue 
to  complete  hydrolysis,  if  appropriate  conditions  were  present ;  we  shall 
see  later  that  an  enzyme-action  comes  to  an  end-  or  equilibrium-point 
owing  to  the  accumulation  of  the  products  of  the  reaction,  so  that  by  dilu- 
tion, or  removal  of  the  products,  the  reaction  can  be  caused  to  go  on 
farther.  The  final  part  of  the  change  may  be  very  slow,  so  that 
very  prolonged  periods  of  action  may  be  necessary  to  detect  it;  the 
case  of  pepsin  is  instructive  in  this  connection ;  it  was  thought  until  re- 


ENZYMES  AS  CATALYSTS  9 

cently  that  this  enzyme  was  unable  to  hydrolyse  proteins  beyond  the 
stage  of  "  peptones  "  ;  we  know  now  that,  if  sufficient  time  be  allowed, 
amino-acids  are  produced.1  The  formation  of  alcohol  from  sugar  is  an- 
other difficult  case.  It  appears  that  different  enzymes  produce  different 
bodies  from  the  same  substrate.  The  weight  falling  along  an  inclined 
plane,  as  used  for  an  illustration  in  the  preceding  chapter,  gives  us  a 
hint  here ;  it  was  pointed  out  that,  although  the  total  energy  change 
was  the  same  whether  much  or  little  catalyst  (oil)  was  present,  yet  the 
form  of  energy  might  be  quite  different,  in  the  one  case  heat,  in  the 
other  kinetic  energy. 

Notwithstanding  what  has  been  said,  the  fact  remains  as  yet  not 
satisfactorily  explained  why  one  enzyme  effects  part  of  a  change  as 
rapidly  as  another  enzyme,  for  example,  the  production  of  peptones  by 
pepsin  and  trypsin ;  while  amino-acids  are  rapidly  formed  by  the  latter 
enzyme  and  but  very  slowly  by  the  former. 

Were  it  not  that  it  seems  impossible  to  place  enzymes  in  any  other 
category  than  that  of  catalysts,  the  anomalies  above  touched  upon 
would  be  more  serious.  It  is  quite  evident  that  chemical  reactions  in 
combining  proportion  are  out  of  the  question,  since  the  enzyme  is  not  a 
constituent  of  the  final  products  and  the  amount  of  these  is  independent 
of  the  amount  of  enzyme  added.  Moreover,  anything  of  the  nature  of 
"trigger  action"  is  excluded  by  the  fact,  familiar  to  all  who  have  made 
experiments  with  enzymes,  viz^  that  the  effect  as  regardsL-tlie  .rate  of, 
action  is  directly  proportional  to  the  concentration  of  the  enzyme. 

On  the  whole,  considering  how  little  we  know  as  yet  about  the  inti- 
mate nature  of  catalytic  phenomena  in  general,  there  is  no  doubt  that 
the  difficulties  referred  to  will  sooner  or  later  be  removed. 

An  interesting  fact  bearing  on  the  subject  before  us  was  discovered 
by  Aders  Plimmer  and  myself  (134,  p.  457)  when  working  at  the 
manner  in  which  trypsin  separates  the  phosphorus  from  caseinogen. 
In  two  months  the  enzyme  separated  only  35  per  cent,  as  inorganic 
phosphate,  whereas  I  per  cent,  caustic  soda  separated  off  the  whole  in 
twenty-four  hours,  leaving  behind  a  body  differing  comparatively  little 
from  the  original  caseinogen.  This  result,  combined  with  the  fact  that 
the  60  per  cent,  left  in  an  organic  form  by  trypsin  was  not  decomposed 
by  subsequent  action  of  alkali  into  inorganic  phosphates,  shows  that 
the  action  of  trypsin  does  not  consist  in  the  activation  of  hydroxidion, 
as  has  been  suggested,  but  that  the  enzyme  acts  as  a  specific  catalyst. 

The  next  point  to  examine  is  the  behaviour  of  enzymes  with  re- 
spect to  the  final  result.  We  have  seen  that  in  catalysis  this  is  inde- 
pendent of  the  quantity  of  catalyst  present,  provided  that  the  latter  has 
not  disappeared  from  the  sphere  of  action  before  the  end-point  is 
reached.  Fig.  I  will  serve  to  show  this  in  the  case  of  trypsin.  The 
series  of  curves  represent  the  increase  in  electrical  conductivity  in  a  5 
per  cent,  caseinogen  solution  under  the  action  of  various  relative 
amounts  of  the  same  enzyme  preparation,  as  marked  on  the  curves. 
The  changes  in  conductivity  are,  as  I  have  shown  (16),  proportional  to 
the  amount  of  peptone  and  amino-acids  produced.  Several  things  are 
to  be  noticed  in  these  curves.  The  rate  of  the  change,  up  to  about  the 
eightieth  minute,  is  directly  proportional  to  the  concentration  of  the 

1  See  Note  B. 


10 


THE  NATURE  OF  ENZYME-ACTION 


CHANCE  IN  CEMMHOS 


FIG.  i. 


ENZYMES  AS  CATALYSTS 


ii 


enzyme,  as  seen  in  the  relative  steepness  of  the  curves.  As  the  reac- 
tion goes  on,  one  curve  after  another  joins  that  of  the  larger  amount, 
until  finally  they  all  arrive  at  the  same  position.  The  one  with  the  least 
amount  of  enzyme  did  not  arrive  at  this  height  during  the  time  shown 
in  the  curve,  probably  because  of  the  destruction  of  the  trypsin,  which 
is  an  unstable  body.  The  last  fact  shows  the  necessity  of  care  in  inter- 
preting the  results  of  experiments  made  for  the  purpose  of  tracing  the 
relation  between  concentration  of  enzyme  and  effect  produced.  It  was, 
at  one  time,  denied  by  some  observers  that  Buchner's  zymase,  the 
alcohol-producing  body  of  yeast,  had  the  characters  of  an  enzyme. 
But  if  the  tables  on  pp.  160-62  of  Buchner's  Zymasegarung  (41)  be 
consulted,  it  will  be  seen  that  its  behaviour  closely  resembles  that  of 
trypsin  ;  it  is  probable  that  the  zymase  is  destroyed  by  the  proteoclastic 
enzyme  present  with  it  in  the  yeast-juice. 

Many  enzymes,  such  as  amylase,  can  be  found  at  the  end  of  the  re-  • 
action  apparently  unaltered  (ErTront  [54,  p.  155]),  and  can  then  act 
upon  a  further  supply  of  substrate.  Even  trypsin,  when  in  presence  of 
a  high  concentration  of  substrate  or  products,  is  not  at  all  rapidly  de- 
stroyed. This  circumstance  is  sometimes  made  use  of  in  preparing 
enzymes  from  the  cells  in  which  they  occur,  e.g.,  pepsin  from  gastric 
mucous  membrane,  by  allowing  it  to  digest  itself  in  presence  of  acid  at 
40°  C. 

The  fact  that  a  small  amount  of  enzyme  is  as  equally  effective  as  a 
larger,  if  sufficient  time  be  allowed,  was  made  use  of  by  the  author  in 
conjunction  with  Starling  to  decide  the  dispute  as  to  the  nature  of  en- 
terokinase  (19).  This  body,  discovered  by  Chepovalnikoff  in  the  Suc- 
cus  entericusy  has  the  power  of  converting  the  inactive  zymogen  of  the 
pancreatic  juice,  as  secreted,  into  active  trypsin.  In  the  opinion  of 
Pavloff,  the  body  has  the  nature  of  an  enzyme,  whereas  Delezenne  and 
others  regard  the  action  as  consisting  in  the  chemical  union  of  the  two 
bodies  to  form  the  third,  trypsin.  If  this  latter  view  be  correct,  the 
quantity  of  trypsin  formed  will  be  in  exact  proportion  to  the  amount  of 
enterokinase  used.  We  found,  on  the  contrary,  that  quantities  of  the 
enterokinase  solution,  varying  from  O'OOOi  c.c.  to  r6  c.c.  per  10  c.c.  of 
juice,  if  allowed  to  act  for  two  days,  produced  the  same  amount  of  tryp- 
sin, within  the  limits  of  experimental  error.  In  using  the  method  for 
such  a  purpose  it  is  obviously  essential  to  take  care  that  the  least 
amount  added  is  not  large  enough  to  combine  with  the  whole  of  the 
body  to  be  changed,  otherwise  there  would,  even  in  case  of  a  true 
chemical  compound,  be  no  difference  between  the  action  of  large  and 
small  amounts.  The  kind  of  results  obtained  in  an  experiment  of  the 
kind  mentioned  are  seen  in  the  table  below. 

The  first  column  gives  the  quantity  of  enterokinase  solution  in  ir6 
c.c.  of  secretin  juice,  the  other  three  the  length  in  mm.  of  gelatin  of 
Mett's  tubes  digested  in  the  time  noted  at  the  head  of  the  column. 


Enterokinase  added 
c.c. 

In  first  six 
hours. 

In  next  eighteen 
hours. 

In  nexf  twenty- 
four  hours. 

O'l 

0-4 
1-6 

o 

2 

3 

10 

ii 

10-5 
9-25 
9 

12  THE  NATURE  OF  ENZYME-ACTION 

Owing  to  the  absence  of  appreciable  quantities  of  substrate  or  products 
a  slight  destruction  of  trypsin  had  taken  place  in  the  cases  where  it  had 
been  formed  at  an  early  stage. 

Instances  were  given  in  the  previous  chapter  of  the  minute  traces  in 
which  catalysts  are  able  to  exercise  their  action.  This  is,  of  course,  not 
surprising  when  the  catalyst  remains  unchanged  at  the  end  of  its  work  ; 
at  the  same  time  it  is  perhaps  striking  that  such  a  very  small  concen- 
tration should  have  any  effect  whatever,  no  matter  how  long  it  were 
allowed  to  act.  In  the  case  of  enzymes  we  find  similar  facts.  Invertase, 
according  to  O'Sullivan  and  Tompson  (129),  can  hydrolyse  200,000  times 
its  weight  of  saccharose ;  rennet,  according  to  Hammarsten,  can  clot 
400,000  times  its  weight  of  caseinogen  in  milk,  and  so  on.  When  we 
remember  that  these  preparations  consist,  in  all  probability,  only  to  a 
small  extent  of  the  actual  enzyme,  their  activity  becomes  all  the  more 
astonishing. 

Since,  as  we  shall  see  in  more  detail  in  a  later  chapter,  there  are  good 
reasons  for  the  statement  that  reactions  catalysed  by  enzymes  are,  as  a 
general  rule,  reversible  ones,  it  is  of  considerable  importance  to  know 
whether  those  which  are  known  to  accelerate  a  decomposition  process 
also  accelerate  the  opposite  or  synthetic  process,  just  as  we  have  seen 
in  the  case  of  inorganic  catalysts  such  as  hydrion.  The  question  will 
come  up  for  discussion  in  a  subsequent  chapter,  so  that  I  will  merely 
state  here  that  it  has  been  shown  for  many  enzymes  that  such  is  the 
case.  Maltase  forms  glucose  from  maltose  and  a  disaccharide  from 
glucose,  emulsin  hydrolyses  salicin  and  also,  under  different  conditions, 
synthesises  it  from  glucose  and  saligenin,  lipase  accelerates  the  hydrolysis 
of  ethyl  butyrate  and  also  its  formation  from  butyric  acid  and  ethyl 
alcohol,  and  so  on.1 

.  As  to  an  influence  on  the  position  of  equilibrium,  we  have  seen  that, 
if  the  catalyst  acts  by  the  formation  of  intermediate  combinations,  this 
position  is  not  necessarily  the  same  as  that  of  the  uncatalysed  reaction, 
or  even  that  attained  under  the  influence  of  a  different  catalyst.  Now, 
since  enzymes  act  by  forming  intermediate  combinations,  of  what  kind 
will  be  discussed  later,  it  is  not  surprising  to  find  that  the  equilibrium 
position  under  lipase  is  not  the  same  as  that  under  hydrion  (Dietz,  [48, 
p.  320]).  Visser  (164,  p.  301)  has  shown  that  in  the  cases  of  invertase 
and  emulsin  the  concentration  of  the  enzyme  has  no  effect  on  the  posi- 
tion of  equilibrium. 

~  Unlike  inorganic  catalysts,  enzymes  are  destroyed  by  temperatures 
from  60°  to  100°,  differing  according  to  the  conditions  present.  This 
behaviour,  though  of  practical  importance,  does  not  affect  their  claim  to 
be  regarded  as  catalysts  and  will  be  explained  in  the  next  chapter. 
The  phenomenon  of  an  apparent  optimum  temperature,  in  which 
enzymes  differ  from  the  majority  of  other  catalysts,  will  also  be  ex- 
plained later  and  shown  to  be  due  to  injury  by  raised  temperature. 

1  Note  C. 


CHAPTER  III. 

CHEMICAL  AND  PHYSICAL  PROPERTIES  OF  ENZYMES. 

ONE  of  the  most  important  physical  properties  of  enzymes  is  their  col- 
loidal nature,  as  shown  by  the  fact  that  they  do  not  pass  through  parch- 
ment-paper, or  do  so  with  extreme  slowness.  Since  many  other 
characteristics  are  dependent  on  this  fact,  it  is  advisable  to  devote  a 
little  space  to  the  discussion  of  the  essential  properties  of  colloidal  solu- 
tions in  so  far  as  they  are  of  importance  with  respect  to  the  nature  and 
mode  of  action  of  enzymes. 

The  first  thing  that  strikes  an  observer  as  regards  colloidal  solutions 
is  that  bodies  which  in  the  usual  sense  of  the  word  are  insoluble  in  water, 
metallic  platinum  or  arsenious  sulphide,  for  example,  can,  by  appropriate 
methods,  be  induced  to  "  dissolve  "  in  water  so  as  to  form  a  permanent 
solution,  which  in  many  ways  behaves  differently  from  a  true  solution. 
By  various  means  it  can  be  shown  that  the  body  so  taken  up  in  water 
is,  in  reality,  in  the  state  of  extremely  minute,  "ultra-microscopic"  par- 
ticles, that  is,  particles  too  small  to  be  visible  under  ordinary  methods 
of  illumination  of  objects  under  the  microscope.1  The  chief  proof  that 
we  have  to  do  with  matter  in  a  particulate  condition  consists  in  the 
application  of  the  Faraday-Tyndall  test.  This  consists  in  passing  a 
powerful  beam  of  light  through  the  liquid  ;  if  particles  be  present,  the 
path  of  the  beam  can  be  readily  seen  as  a  bright  streak,  when  looked  at 
from  the  side.  In  the  case  of  permanent  colloidal  solutions,  which  do 
not  deposit  on  standing,  the  light  sent  out  at  right  angles  to  the  beam  is 
found  to  be  polarised ;  this  means  that  the  particles  reflecting  it  are 
smaller  than  the  mean  wave-length  of  the  light  forming  the  beam. 

If  now  we  consider  in  what  respect  the  particles,  say,  in  a  colloidal 
solution  of  platinum  prepared  by  Bredig's  method  of  an  arc  between 
platinum  electrodes  under  water  (35,  p.  265),  differ  from  an  equal  amount 
of  the  metal  in  the  form  of  sheet  or  wire  immersed  in  water,  it  is  plain 
that  it  consists  in  the  relatively  enormous  development  of  surface  in  the 
former  case.  According  to  Siedentopf  and  Zsigmondy  (172)  the  par- 
ticles in  certain  colloidal  solutions  of  gold  had  a  radius  of  one-millionth  of 
a  centimetre  ;  a  sphere  of  gold  with  a  radius  of  I  millimetre  if  divided 
into  particles  of  these  dimensions  would  have  a  surface  of  some  100 
square  metres. 

It  might,  perhaps,  be  supposed  that  if  the  process  of  subdivision 
were  carried  farther  and  farther  until  molecular  dimensions  were 
reached,  the  phenomena  due  to  surface  would  be  more  and  more 

1  It  is  pointed  out  by  Hardy  (77,  p.  no)  that,  according  to  the  phase- rule,  these  col- 
loidal solutions  probably  consist  of  suspensions  of  solid  particles,  containing  a  small  amount 
of  water,  in  a  dilute  true  solution  of  the  solid  body  in  water.  These  are  the  two  phases  of 
the  heterogeneous  system.  The  same  view  is  taken  by  Quincke  (184,  p.  1012). 


14  THE  NATURE  OF  ENZYME-ACTION 

manifest.  In  point  of  fact,  however,  this  is  not  the  case ;  although  we 
know  nothing  of  what  the  molecular  state  essentially  is,  we  do  know 
that  bodies  in  this  condition  do  not  show  the  properties  of  matter  in 
mass,  z>.,  bounded  by  surfaces.  At  the  same  time  it  must  be  under- 
stood that  there  is  no  hard  and  fast  line  to  be  drawn  between  matter  in 
pieces  visible  to  the  naked  eye,  down  through  ultra-microscopic  par- 
ticles to  molecules.  Such  properties  as  osmotic  pressure,  diffusibility, 
etc.,  are  exhibited  by  all  and  in  proportion  to  the  dimensions  of  their 
elements.  Certain  of  these  properties  are  more  pronounced  in  some 
of  the  above  stages,  others  in  other  stages.  For  example,  osmotic  pres- 
sure, which  is  a  function  of  the  number  of  bodies  acting  as  individuals, 
whether  molecules,  ions  or  particles,  in  unit  volume,  will  naturally  be 
greater,  the  greater  the  number  of  bits  a  given  mass  is  divided  into ; 
whereas  the  phenomena  due  to  surface  can  only  be  present  as  long  as 
the  particles  still  have  the  properties  of  matter  in  mass. 

There  is  one  respect  in  which  a  real  distinction  seems  to  exist.  An 
atom  or  molecule,  when  it  has  an  electric  charge,  is  called  an  ion ;  col- 
loidal particles  are  also  capable  of  carrying  a  charge,  but  whereas  in  the 
former  case  this  charge  is  invariable  in  amount  and  always  the  same  in 
sign  for  each  kind  of  ion,  in  the  latter  case  the  charge  is  variable,  both 
in  sign  and  quantity.  The  probable  reason  for  this  j\ve  shall  see  pre- 
sently. 

VThe  great  distinguishing  characteristic  pf  colloids,  accordingly,  is 
the  enormous  development  of  surface.  We  must,  therefore,  look  for 
properties  dependent  on  this.  Now  the  most  salient  of  these  is  that  of 
^-gurface-tension.  When  solids  are  immersed  in  fluids  the  film  at  the 
interface  where  the  two  are  in  contact  behaves  as  if  stretched.  Any 
free  surface  of  a  liquid  shows  this  property.  Its  real  existence  can 
easily  be  made  manifest  by  taking  a  wire  ring  with  a  thread  tied  to  two 
points  so  as  to  lie  loosely  across  the  space ;  the  ring  is  then  dipped  into 
soap  solution  so  as  to  make  a  film  in  which  the  thread  floats ;  if  the 
film  be  now  broken  on  one  side  of  the  thread  by  touching  it  with  a 
pointed  bit  of  filter-paper,  the  thread  is  immediately  drawn  into  the  arc 
of  a  circle  by  the  tension  of  the  rest  of  the  film.  Suppose,  then,  that 
the  colloidal  particles  are  suspended  in  a  liquid  containing  in  solution  a 
substance  which  lowers  the  surface-tension  of  the  liquid,  as  in  fact  the 
majority  of  bodies  do,  it  is  plain  that,  if  this  substance  accumulates  at 
the  interface  of  the  solid  and  liquid,  the  surface-tension  at  this  place 
will  be  lowered.  It  can  be  shown  thermodynamically  that  such  a  pro- 
cess tending  to  a  diminution  of  tension  will  of  necessity  occur  (Willard 
Gibbs  [189]).  It  is,  in  fact,  the  phenomenon  known  as  "adsorption,"  which- 
plays  so  large  a  part  in  the  behaviour  of  colloids,  and  everywhere  where 
surfaces  are  in  contact  with  liquids  or  gases.  The  phenomenon  can 
readily  be  seen  by  taking  a  dilute  solution  of  a  dye  such  as  congo-red 
and  immersing  a  piece  of  filter-paper  in  it ;  after  a  time  the  dye  will  be 
seen  to  have  concentrated  on  the  paper,  and,  if  the  solution  was  not  too 
strong,  the  colour  will  have  nearly  vanished  from  the  solution  and  be 
transferred  to  the  paper.  Such  a  process  is  obviously  of  much  import- 
ance in  cell-activity,  where  the  constituents  of  the  protoplasm  are  col- 
loids and  the  various  bodies  which  arrive  to  be  acted  upon,  or  themselves 
to  act,  are  in  very  dilute  solution.  It  also  plays  a  prominent  part  in 
enzyme-action,  as  will  often  be  seen  in  subsequent  pages. 


CHEMICAL  PROPERTIES  OF  ENZYMES  15 

There  are  some  properties  of  colloids  which  need  a  little  more  de- 
tailed discussion  in  respect  to  their  connection  with  the  problem  before  us. 

In  the  first  place  there  is  diffusibility.  The  fact  that,  as  definitely 
shown  by  Starling  (147,  p.  323,  and  148,  p.  318)  and  by  Moore  and  Roaf 
(i 21),  colloids  have  a  measurable,  though  small,  osmotic  pressure 
proves  that  their  particles  possess  that  amount  of  motion  which  is 
necessary  to  cause  an  osmotic  pressure.  They  are,  therefore,  not  abso- 
lutely indiffusible.  Haemoglobin  is,  accordingly,  found  to  diffuse 
slowly  into  a  jelly  of  gelatin.  As  to  their  ability  to  pass  through  mem- 
branes, this  is  a  question  entirely  of  the  structure  of  the  membrane, 
especially  as  to  the  dimensions  of  its  pores.  Congo-red,  which  shows 
all  the  usual  characters  of  colloids,  will  pass  slowly  through  some 
samples  of  parchment-paper,  but  not  at  all  through  others.  The  ani- 
line dyes  manifest  all  possible  degrees  of  diffusibility,  in  accordance 
with  the  dimensions  of  their  ultra-microscopic  particles.  Similarly, 
although  most  enzymes  do  not  diffuse  through  parchment- paper,  it 
appears  that  invertase  does  so  to  a  slight  degree,  and  "diastase,"  as 
prepared  by  Fraenkel  and  Hamburg  (67,  p.  396)  from  malt,  although 
showing  an  illuminated  cone  in  the  ultra-microscope  and  therefore 
containing  particles,  which  were  too  small  to  be  seen  separately,  was 
found  to  be  divided  by  dialysis  into  two  distinct  enzymes,  one  of  which, 
viz.)  that  which  caused  the  formation  of  sugar,  passed  through  the 
paper,  while  the  other,  which  liquefied  starch,  was  left  behind. 

In  the  second  place,  colloidal  solutions  exhibit  the  phenomena 
known  as  "  hysteresis  ".  While  a  sodium  chloride  solution  is  always  the 
same  whatever  its  previous  history,  frozen,  boiled  or  kept  for  any  length 
of  time,  colloidal  solutions  are  not  the  same  after  experiencing  any 
of  these  actions.  In  other  words,  they  are  unstable  and  liable  to 
spontaneous  change.  Thus,  enzymes  in  solution'  lose  their  activity 
more  or  less  rapidly.  Many,  like  trypsin,  slowly  lose  activity  even  in 
the  air-dry  state,  and  can  only  be  kept  for  any  length  of  time  in  a  desic- 
cator in  a  cool  and  dark  place. 

In  the  third  place  very  many  colloids,  both  organic  and  inorganic, 
possess  naturally  an  electrical  charge,  positive  or  negative,  and  those 
which  have  not  such  a  charge  can  be  caused  to  take  up  one  by  the  action 
of  electrolytes.  Arsenious  sulphide,  as  prepared  by  passing  hydrogen 
sulphide  through  a  solution  of  arsenious  acid,  has  a  negative  charge, 
while  ferric  hydroxide,  prepared  by  the  dialysis  of  a  ferric  chloride  solu- 
tion, has  a  positive  charge  ;  congo-red  has  a  negative  charge,  night-blue 
a  positive  one ;  serum-globulin,  in  neutral  solution,  has  no  charge,  in  acid 
solution  a  positive  one,  in  alkaline  solutions  a  negative  one.  In  this 
latter  case  there  seems  no  doubt  that  the  colloid  takes  up  hydrions  or 
hydroxidions  from  the  solution  and  so  obtains  its  charge.  The  precise 
cause  of  the  charge  in  other  cases  is  not  clear.  Most  solid  bodies  in 
suspension  in  water  have  a  negative  charge,  as  shown  by  Quincke,  but 
this  charge  can  be  reversed  in  sign  by  addition  of  acid  (Perrin  [132]). 
The  existence  of  a  charge  can  be  detected  by  exposing  the  solution  to 
an  electric  field  between  electrodes  in  a  tube,  and  using  preferably  Whet- 
ham's  boundary  method  (167,  pp.  342-45);  the  colloid  particles  will 
travel  towards  the  pole  of  opposite  sign  to  that  of  their  own  charge  (see 
the  investigations  of  Hardy,  74,  75  and  76). 

Statements  have  been  made  to  the  effect  that  enzymes  are  electrically 


* 

16  THE  NATURE  OF  ENZYME-ACTION 

charged.  Probable  as  this  is,  it  is  perhaps  premature  to  accept  it  until 
we  know  that  we  have  pur£  enzymes  in  our  hands. 

As  we  have  seen,  colloids  take  up  by  adsorption  various  other  bodies, 
and  especially  colloids.  Although  this  process  occurs  when  either  or 
both  of  the  bodies  concerned  is  uncharged,  it  is  naturally  greatly  in- 
creased or  diminished  if  they  have  opposite  or  similar  charges  respect- 
ively. These  "adsorption-compounds,"  or  "colloidal-complexes,"  as 
they  are  called  by  some  when  both  constituents  are  colloids,  play  a  very 
important  part  in  enzyme-action  and  in  fact  in  physiological  chemistry 
generally.  To  understand  their  nature,  or  rather  their  chief  properties, 
let  us  take  a  concrete  case.  When  electro-negative  arsenious  sulphide 
is  mixed  with  electro-positive  ferric  hydroxide  in  such  proportions  that 
the  resulting  mixture  is  electrically  neutral,  both  bodies  are  precipitated 
in  the  form  of  a  complex  colloid,  and  the  fluid  becomes  clear  and  colour- 
less. It  is  sometimes  stated  that  this  precipitate  is  soluble  in  excess  of 
either  colloid.  If  we  make  mixtures  of  these  in  series,  beginning 
with  excess  of  the  one  and  ending  with  excess  of  the  other,  we  shall  see 
that  the  statement  does  not  correctly  express  the  state  of  affairs.  Sup-* 
pose  that  we  have  so  arranged  the  series  that  the  mixture  in  electrical 
neutrality  and  total  precipitation  is  in  the  middle,  we  notice  that  in  all 
the  various  mixtures  there  is  a  precipitate,  but  that  it  progressively 
diminishes  as  we  proceed  towards  either  end,  />.,  as  the  excess  of  one 
or  of  the  other  colloid  becomes  greater.  Further,  since  the  two  colloids 
used  are  conveniently  of  different  colours,  it  is  easy  to  see  by  their  colour 
that  all  ithe  precipitates  contain  both  colloids,  but  in  varying  relative 
proportion,  according  to  the  excess  of  either  colloid  in  the  mixture.  The 
same  statement  is  to  be  made  with  respect  to  the  supernatant  fluids,  all 
of  which  contain  both  colloids.  A  series  of  new  colloids  has  therefore 
made  its  appearance,  consisting  of  adsorption-compounds  of  the  original 
colloids  in  all  possible  proportions.  These  compounds  differ  from  those 
of  pure  chemistry  in  that,  instead  of  being  in  constant  combining  pro- 
portion, they  are  compounds  in  varying  combining  proportion,1  the 
proportion  being  determined  by  the  relative  concentration  of  the  two 
colloids  in  the  mixture. 

An  important  characteristic  of  adsorption  phenomena  is  the  form  of 
the  law  which  governs  the  composition  of  the  compounds.  To  under- 
stand this  expression  it  is  best  to  take  a  concrete  case  again.  Picric 
acid  is  soluble  in  water  and  in  ether,  but  it  is  more  soluble  in  ether  than 
in  water;  with  constant  quantities  of  water,  ether  and  picric  acid  a 
definite  fraction  of  the  latter  will  be  present  in  the  ether,  say  four-fifths ; 
let  us  now  double  the  amount  of  picric  acid,  the  proportion  of  solvents 
remaining  the  same  as  before  ;  as  is  familiar  to  all,  the  amount  of  picric 
acid  both  in  the  ether,  and  in  the  water,  will  be  doubled.  This  is  an 
instance  of  a  physical  partition  in  proportion  to  relative  solubility.  Take 
now  a  case  of  a  purely  chemical  reaction  such  as  silver  nitrate  and 
hydrochloric  acid  ;  if  to  a  solution  of  silver  nitrate  sufficient  hydrochloric 
acid  to  combine  with  the  silver  be  added,  the  whole  of  the  latter  will  be 
precipitated,  so  that  the  addition  of  more  hydrochloric  acid  will  bring 
down  no  further  quantity.  Take  finally  a  case  of  an  adsorption  re- 
action between  colloids,  cellulose  in  the  form  of  filter-paper,  which  acts 

iSee'Qstwald  (127,  i.,  p.  1097). 


CHEMICAL  PROPERTIES  OF  ENZYMES  17 

precisely  like  a  colloid,  and  a  dilute  solution  of  congo-red.  After  a 
certain  time  it  will  be  found  that  part  of  the  dye  has  deposited  itself 
on  the  paper,  but  not  the  whole ;  that  is,  the  reaction  partakes  of  the 
nature  of  both  the  physical  and  chemical  reactions ;  physical,  in  that 
there  is  dye  both  in  the  paper  and  in  the  solution,  chemical,  in  that,  like 
the  silver  chloride,  there  is  a  kind  of  precipitation.  If  we  double  the 
concentration  of  the  dye  we  shall  find  that,  unlike  the  chemical  reaction, 
more  congo-red  is  deposited  on  the  paper ;  and  unlike  the  physical  process, 
it  will  be  found,  on  making  quantitative  experiments,  that  the  amount  of 
dye  taken  up  is  not  doubled,  but  something  less,  in  fact  multiplied  by 
some  root  of  2  ;  in  most  adsorption  processes  this  root  is  found  to  be  less 
than  the  square  root.  Expressed  in  algebraic  form,  when  the  concentra- 
tion of  the  solution  is  doubled  the  amount  adsorbed  is  not  multiplied  by  2, 

but  by  2  to  the  power  -  where  n  is  greater  than  I,  and  usually  less  than 

2,  viz.,  from  1-4  to  17.  It  is  obvious,  however,  that  we  are  justified  in 
applying  the  name  of  adsorption  to  cases  of  any  values  of  ny  less  than 

infinity,1  in  which  case  the  reaction  is  purely  chemical ;  —  here  becomes 

o,  and  all  numbers  to  the  power  o  become  unity ;  in  other  words,  the 
amount  of  silver  chloride  in  the  example  above  is  always  the  same 
whatever  the  concentration  of  the  hydrochloric  acid.  When  n  =  i  it  is 
plain  that  the  physical  process  of  partition  according  to  solubility  is 
expressed. 

We  see  then  that  the  important  point  about  the  process  of  adsorption 
is  that  relatively  more  of  the  compound  is  formed  in  the  more  dilute 
solutions.  Many  important  facts  in  the  chemistry  of  colloids,  especially 
in  the  relation  of  toxin  to  antitoxin,  find  their  explanation  in  this  cir- 
cumstance. 

It  is  of  interest  to  consider  for  a  moment  what  the  expression  above 
given  means  when  n  is  less  than  I  ;  it  must  refer  to  cases  where  relatively 
more  of  the  adsorption-comgpund  is  formed  in  the  stronger  solutions. 
Although  such  cases  are  rare,  it  appears  that  they  may  occur. 

It  will  readily  be  understood  that,  although  adsorption  can  take 
place  between  uncharged  colloids,  or  even  those  of  similar  charge,  the 
process  is  much  facilitated  when  the  bodies  have  charges  of  opposite 
sign.  The  fact  is  well  shown  by  the  relation  of  paper  to  colloidal  dyes. 
In  water,  paper  has  a  negative  charge,2  congo-red  also  is  electro-negative, 
accordingly,  there  is  practically  no  adsorption.  Night-blue,  on  the  other 
hand,  is  electro-positive,  so  that  it  is  adsorbed  in  large  amount  (14,  p. 
205). 

Before  proceeding  to  the  subject  of  the  influence  of  electrolytes  on 
colloids,  we  may  briefly  refer  to  the  causes  of  the  permanency  of  col- 
loidal suspensions.  There  are  several  causes  which  have  been  suggested 
and,  no  doubt,  in  many  cases  these  co-operate.  We  have  seen  how 
neutralisation  of  electric  charges  results  in  precipitation  in  some  cases, 
hence  it  was  suggested  by  Hardy  (77,  p.  114)  that  mutual  repulsion  of 
similarly  charged  particles  was  in  some  cases  responsible*  for  the  per- 

1Freundlich  (175),  who  has  done  much  important  work  on  this  subject,  is  frequently 
understood  as  limiting  the  title  of  "  adsorption  "  to  reactions  with  particular  values  of  this 
exponent.  It  does  not  appear  from  his  work  that  he  intended  to  do  this. 

2  See  Winkelmann,  Handbuch  der  Physik,  2te  Auflage,  1905,  p.  955. 

2 


18  THE  NATURE  OF  ENZYME-ACTION 

manency  of  the  solution.  But  there  are  many  uncharged  colloids  equally 
permanent,  so  that  there  must  be  some  other  reason.  Probably  the  very 
minuteness  of  the  particles  prevents  their  deposition,  owing  to  the  re- 
latively great  frictional  resistance ;  the  fact  that  these  particles  are  in 
continual  motion,  like  those  of  gases,  must  also  be  remembered.  This 
is  shown  by  their  osmotic  pressure,  as  also  by  the  fact  that  they  can 
diffuse  in  opposition  to  gravity,  as  can  be  seen  by  haemoglobin  passing 
into  a  tube  of  solidified  gelatine  suspended  mouth  downwards  in  a  solu- 
tion of  this  body. 

We  have  seen  that  an  electrically  charged  colloid  is  able  to  throw 
down  a  colloid  which  has  a  charge  of  the  opposite  sign  ;  it  is  therefore 
/not  surprising  to  find  that  electrolytes  have  the  same  kind  of  effect. 
/  Arsenious  sulphide,  being  negative,  is  precipitated  by  positive  ions. 
Ferric  hydroxide,  being  positive,  is  precipitated  by  negative  ions.  Now 
of  course  in  adding  an  electrolyte,  sodium  chloride,  for  example,  we  are 
adding  both  positive  and  negative  ions,  which  will  have  opposite  effects. 
In  point  of  fact,  in  these  comparatively  simple  cases  the  ion  which  pre- 
cipitates is  found  to  be  prepotent.  How  do  we  know  that  it  is  the  one 
of  opposite  sign  ?  The  answer  is  by  comparing  the  effects  of  series  of 
salts  with  common  kation  or  anion  respectively.  The  chloride,  sulphate 
and  phosphate  of  sodium  are  nearly  equal  in  action  on  negative  colloids, 
whereas  on  positive  colloids  the  effect  of  the  sulphate  is  considerably 
more  than  double  that  of  the  chloride,  and  that  of  the  phosphate  very 
much  more  than  three  times. 

Organic  colloids,  e.g.,  serum-globulin,  can  be  made  either  positive  or 
negative  by  acid  or  alkali  respectively,  as  previously  stated.  When 
positive  they  are  precipitated  by  negative  colloids  or  ions,  such  as  ar- 
senious  sulphide  or  the  ferrocyanic  ion.  When  negative,  positive  colloids 
or  ions  are  the  active  agents. 

The  possibilities  when  mixtures  of  various  colloids  and  ions  occur, 
as  in  the  living  cell,  will  thus  be  seen  to  be  extremely  complex,  indeed, 
as  yet  but  little  is  definitely  known  of  them.  The  case  of  two  colloids 
and  an  electrolyte  is  comparatively  simple  and  may  be  mentioned. 
Paper  and  congo-red,  which  are  both  negative,  have  but  little  adsorptive 
affinity  for  each  other ;  the  presence  of  positive  ions,  however,  causes  a 
large  adsorption  to  take  place,  while  anions  have  very  little  effect.  Unlike 
what  happens  in  this  comparatively  simple  case,  if  we  take  negative 
paper  and  positive  dye,  the  action  of  an  electrolyte  is  also  that  of  the 
kation ;  so  that  less  adsorption  takes  place  than  in  the  absence  of  the 
electrolyte  (14,  p.  203). 

It  is  very  important  to  bear  in  mind  that  all  these  colloidal  reactions 
have  both  a  physical  and  chemical  aspect.  Certain  colloids  have  a 
special  adsorptive  affinity  for  one  another,  which  is  not  purely  chemical, 
since  it  follows  the  laws  of  adsorption  and  not  those  of  constant  com- 
bining proportion.  This  fact  is  of  great  importance  with  regard  to 
enzymes. 

To  avoid  misconception  attention  must  be  called  to  the  fact  that,  as 
will  probably  have  been  noticed,  many  reactions  can  equally  well  be 
described  in  the  language  of  pure  chemistry  or  in  that  of  colloidal  or 
physical  chemistry.  These  are  in  no  sense  antagonistic,  but  call  atten- 
tion to  different  aspects  of  the  phenomena.  For  instance,  the  well- 
known  test  for  proteins  with  potassium  ferrocyanide  and  acetic  acid  may 


CHEMICAL  PROPERTIES  OF  ENZYMES  19 

be  stated  to  be  due  to  the  insolubility  in  acid  of  the  compound  of  the 
protein  with  the  ferrocyanide ;  or  it  may  be  explained  by  saying  that 
the  protein  in  neutral  solution,  being  a  colloid  without  electrical  charge 
and  hence  unaffected  by  small  amounts  of  electrolyte,  becomes  capable 
of  precipitation  by  the  quadrivalent  anion  of  potassium  ferrocyanide 
when  itself  made  electro-positive  by  the  action  of  hydrion. 

Enzymes  then  are  colloids,  and  have  the  property  of  carrying  down 
with  them,  by  adsorption,  constituents  of  the  solutions  from  which  they 
are  precipitated.  It  is  not  therefore  to  be  wondered  at  that  amylase  or 
invertase,  as  obtained  in  the  usual  way,  give  carbohydrate  reactions,  nor 
that  pepsin  or  trypsin  give  protein  reactions.  It  is  found,  however, 
that  the  more  the  bodies  are  purified,  the  fewer  characteristic  reactions 
of  any  kind  do  they  show,  and  at  the  same  time  the  more  unstable  do 
they  become.  The  possibility  must  not  be  forgotten  that  a  particular 
enzyme  may  be  similar  in  its  constitution  to  the  substrate  on  which  it 
acts,  since  there  is  such  a  close  connection  between  certain  enzymes 
and  their  own  particular  substrates,  as  we  shall  see  later. 

Perhaps  the  purest  preparations  known  as  yet  are  the  invertase  of 
W.  A.  Osborne(i26),  the  amylase  of  Fraenkel  and  Hamburg  (67),  and  the 
pepsin  of  Pekelharing  (131).  Let  us  see  what  the  properties  of  these 
bodies  are,  leaving  the  method  of  preparation  for  the  next  chapter. 

Osborne's  invertase  gave  none  of  the  protein  reactions,  except  pre- 
cipitation by  copper  sulphate,  lead  acetate  and  phospho-tungstic  acid  ; 
it  gave  Millon's,  the  xanthoproteic  and  biuret  tests  very  faintly.  It 
was,  therefore,  not  protein  in  nature.  On  the  other  hand,  it  was  found 
impossible  to  completely  free  it  from  carbohydrate,  which  was  afterwards 
identified  by  Koelle  (103)  as  mannose.  At  present  it  is  not  possible  to 
express  an  opinion  as  to  whether  this  is  an  essential  component  or  not.  It 
is  suggestive  that  Fraenkel  and  Hamburg's  amylase  also  contained  a  carbo- 
hydrate in  small  amount,  but,  in  this  case,  a  pentose.  It  also  showed 
absence  of  typical  protein  reactions,  that  is,  it  gave  neither  the  biuret  nor 
the  xanthoproteic  reactions,  but  a  faint  indication  of  Millon's  reaction. 

The  invertase  could  not  be  freed  from  ash,  but  the  amount  present 
was  variable.  It  always  contained  nitrogen. 

Moreover,  Beijerinck  (22)  proved  that  an  amylase  was  incapable  of 
replacing  either  carbohydrate  or  nitrogenous  bodies  in  a  nutritive  medium 
for  yeasts  or  bacteria. 

Pekelharing's  pepsin,  in  moderately  concentrated  solution,  gave  the 
majority  of  protein  reactions,  but  contained  no  phosphorus,  thus  dispos- 
ing of  the  view  that  enzymes  are  nucleo-proteins.  The  ash  was  very 
small,  0*1  per  cent.  The  preparation  was  laevo-rotatory  and  very 
active,  viz.,  O'ooi  milligramme  in  6  c.c.  of  0*2  per  cent.  HC1  dissolved 
a  flake  of  fibrin  in  less  than  twenty  hours  at  37°  C.  Perhaps  its  most 
interesting  property  is  that  it  is  relatively  insoluble  in  water,  but  freely 
soluble  in  0*2  per  cent,  hydrochloric  acid. 

On  the  whole  it  appears  that  all  enzymes  have  not  the  same  chemi- 
cal structure,  a  fact  probable  enough  in  itself.  Some  indeed  seem  to 
belong  to  a  class  of  bodies  as  yet  unknown  in  chemical  science,  but 
containing  nitrogen  and  carbohydrate  in  their  molecules. 

When  we  consider  the  way  in  which  definite  chemical  properties 
diminish  more  and  more  as  the  preparations  are  purified,  we  see  a  cer- 
tain degree  of  justification  for  the  view  expressed  by  De  Jager  (93)  and 

2  * 


20  THE  NATURE  OF  ENZYME-ACTION 

by  Arthus  (10),  viz.,  that  enzymes  are  not  chemical  individuals,  but 
that  various  kinds  of  bodies  may  have  conferred  upon  them  properties 
which  cause  them  to  behave  like  enzymes ;  so  that  we  have  to  deal 
with  properties  rather  than  substances.  The  action,  it  is  stated,  can 
even  be  exerted  at  a  distance.  The  experiments  brought  forward  in 
support  of  this  view  are  by  no  means  convincing.  I  have  myself  re- 
peated one  of  these,  but  was  unable  to  obtain  the  result  stated  to 
happen.  The  experiment  is  of  sufficient  interest  to  warrant  descrip- 
tion. A  solution  of  pepsin  in  hydrochloric  acid  is  placed  inside  a  parch- 
ment-paper tube  and  outside  of  this  a  solution  of  hydrochloric  acid  of 
the  same  strength.  Cubes  of  boiled  egg-white  are  then  added  to  both 
solutions  and  the  vessel  left  for  some  days  at  38°.  Pepsin  itself  cannot 
pass  out  of  the  tube,  and  yet,  so  it  was  stated,  the  egg-white  in  the 
outer  fluid  was  digested.  I  found,  on  the  contrary,  that  these  cubes 
were  absolutely  intact,  although  the  fluid  gave  a  biuret  reaction  owing 
to  the  diffusion  of  the  products  of  digestion  of  those  in  the  inside  of  the 
tube.  Possibly  there  may  have  been  minute  holes  in  the  tube  of  the 
original  experiment.  A  somewhat  similar  theory  has  been  recently  pro- 
posed by  Barendrecht  (12),  according  to  which  enzymes  act  as  radio- 
active bodies,  the  chemical  activities  of  enzymes  being  due  to  radiations. 

As  already  pointed  out,  inorganic  catalysts  are  definite  chemical  in- 
dividuals ;  there  is,  therefore,  justification  for  holding  that  organic  cata- 
lysts are  also  bodies  of  definite  composition,  at  all  events  until  stronger 
evidence  has  been  brought  to  the  contrary. 

Enzymes,  in  contradistinction  to  inorganic  catalysts,  are  destroyed 
*Cy  exposure  to  a  temperature  below  100°  C,  but  which  varies  according 
to  conditions;  certain  enzymes  are  more  sensitive  than  others.  This 
property  is  no  doubt  due  to  the  colloidal  nature  of  these  bodies,  and  is 
in  practice  a  useful  means  of  detecting  whether  a  substance  belongs  to 
this  class  of  bodies  or  not.  If  certain  changes  continue  to  show  them- 
selves in  a  system  after  it  has  been  boiled  for  some  time,  it  may  be 
taken  with  confidence  that  they  are  not  due  to  the  presence  of  an 
enzyme  in  it.  The  converse  is  not  universally  true,  since  some  colloidal 
bodies  other  than  enzymes  are  coagulated  or  otherwise  altered  by  heat. 

This  characteristic  of  enzymes  obviously  gives  us  no  help  in  decid- 
ing whether  an  action  is  due  to  an  enzyme  or  to  the  agency  of  living 
cells.  Perhaps  the  distinction  is  at  bottom  one  of  words  only,  but  at 
present  there  are  many  changes  known  to  be  produced  by  living  cells, 
changes  of  a  kind  such  as  no  enzymes  yet  known  are  able  to  effect. 
Moreover,  even  if  all  cell-activities  are  due  to  enzymes,  while  the  cells 
are  growing  there  is  a  growth  of  enzymes  also,  so  that  the  course  of  a 
reaction  would  be  quite  different  in  the  two  cases. 

The  use  of  antiseptics  enables  this  distinction  to  be  made.  The  life 
of  protoplasm  is  impossible  in  the  presence  of  an  amount  of  antiseptic 
which  has  little  or  no  effect  on  enzymes.  At  the  same  time  it  must  be 
kept  in  mind  that  some  enzymes  are  very  sensitive  to  certain  anti- 
septics, such  as  formaldehyde.  On  the  whole  toluene  appears  to  be 
the  least  injurious. 

Filtration  through  porous  clay  or  Berkefeld  filters  serves  in  some 
cases  to  exclude  the  presence  of  living  cells,  but  since  all  colloids  are 
more  or  less  adsorbed  by  surfaces,  considerable  loss  takes  place  until 
the  filter  is  saturated  with  the  enzyme. 


CHAPTER  IV. 

GENERAL  METHODS  OF  PREPARATION  AND  OF  INVESTIGATION. 

IT  is  not  within  the  scope  of  this  work  to  describe  in  detail  the  various 
methods  used  in  the  preparation  of  enzymes;  for  this,  the  original 
memoirs  must  be  consulted.  At  the  same  time  it  will  be  useful  to  indi- 
cate the  general  principles  on  which  these  methods  depend,  especially 
as  they  throw  light  on  the  nature  of  enzymes. 

Since  the  greater  number  are  found  in  cells,  it  is  evident  that  they 
must  be  extracted  by  some  means  or  other.  Certain  enzymes  are  found 
already  in  solution,  so  that  this  operation  is  unnecessary;  such  are 
pepsin  in  gastric  juice,  trypsin  (or  rather  its  zymogen)  in  pancreatic 
juice,  erepsin  in  Succus  entericus  and  ptyalin  in  saliva.  The  juices  of 
certain  fruits,  the  pine-apple  and  that  of  the  Papaw  tree  for  example, 
also  contain  proteoclastic  enzymes. 

When  tissues  or  cells  are  extracted  with  water,  best  saturated  with 
toluene  or  chloroform,  it  is  found  that  some  enzymes  are  extracted, 
others  are  not.  In  other  words,  the  cell  limiting-membrane  is  made 
sufficiently  permeable  by  this  means  to  allow  such  an  enzyme  as  invertase 
to  pass  out ;  but  even  in  this  case  better  yields  are  obtained  if  the 
yeast-cells  be  allowed  to  disintegrate  by  autolysis.  Many  enzymes 
in  fact  cannot  be  obtained  at  all  unless  the  cells  are  disintegrated  ;  such 
are  the  zymase  of  yeast  and  the  various  autolytic  enzymes  of  animal 
tissues.  There  are,  accordingly,  two  chief  classes  of  methods  in  use, 
according  as  the  enzyme  is  "  intracellular  "  or  not. 

When  mere  extraction  suffices  it  is  sometimes  advisable  to  use 
glycerol  or  weak  alcohol  in  place  of  water,  in  order  to  avoid  the  deleteri- 
ous effect  of  prolonged  exposure  to  the  latter ;  such  a  case  is  that  of 
the  extraction  of  trypsin  or  rather  trypsinogen,  from  the  pancreatic 
cells. 

Comparatively  little  disintegration  seems  requisite  in  the  case  of 
the  enzymes  of  the  alimentary  canal,  which  hydrolyse  disaccharides,  since 
short  grinding  with  sand  and  toluene  water  in  a  mortar  suffices  if  the 
mixture  be  allowed  to  stand  for  some  hours.  The  drying  of  yeast  is 
efficacious  for  the  extraction  of  similar  enzymes  from  it,  as  was  found 
by  Emil  Fischer  (63)  and  by  Croft  Hill  (86).  The  yeast  is  spread  out 
in  thin  layers  on  glass  or  porous  plates,  and  dried  as  rapidly  as  possible 
in  a  current  of  warm  and  dry  air ;  when  dry,  the  yeast  may,  with  advan- 
tage, be  heated  to  60°  or  70°,  whereby  the  subsequent  extraction  by  water 
or  weak  alkali  is  facilitated,  probably  by  more  effective  disintegration. 
In  the  dry  state  enzymes  are  much  more  resistant  to  heat  than  in  the 
presence  of  water.  The  dried  yeast  obtained  above  may  be  kept  for  a 
considerable  time  without  losing  its  properties  as  an  enzyme. 

21 


22  THE  NATURE  OF  ENZYME-ACTION 

More  thorough  disintegration  is  necessary  in  other  cases.  There  are 
three  chief  methods  for  doing  this. 

The  first  one  is  that  used  by  Buchner  (41,  p.  58),  who  was  the  earliest 
investigator  to  obtain  the  enzyme  of  alcoholic  fermentation.  It  consists 
in  thorough  grinding  with  sand  and  kieselguhr  in  a  mortar ;  the  dry 
mass,  or  rather  thick  dough,  thus  obtained  is  then  exposed  to  a  pressure 
of  300  atmospheres  in  a  hydraulic  press.  The  kieselguhr  is  necessary, 
not  only  as  a  filter,  but  to  afford  a  kind  of  support  to  the  cell-contents 
so  as  to  enable  the  great  pressure  to  squeeze  out  the  fluid  from  them. 
Of  course  the  great  surface  of  the  kieselguhr  adsorbs  a  considerable  part 
of  the  enzyme,  as  shown  by  the  fact  that  the  press-cake  from  the  first 
operation,  by  rubbing  up  again  with  saline  solution  and  repeating  the 
pressing,  gives  a  further  supply  of  the  enzyme. 

The  second  method  is  that  of  Rowland  (140),  who  used  a  kind  of 
toothed  wheel  kept  in  very  rapid  rotation  in  the  mixture  of  cells  and 
sand.  Apparently  the  grains  of  sand  are  driven  with  great  force  against 
the  cells  and  thus  break  them  to  pieces.  A  modification  of  this  method, 
specially  applicable  to  bacteria,  consists  in  freezing  the  cells  solid  by 
means  of  liquid  air  and  grinding  them  in  this  state  in  a  steel  mortar  by 
means  of  a  machine. 

The  third,  and  most  recent,  method  may  be  described  as  a  devel- 
opment of  the  last.  It  is  due  to  Wiechowski  (169),  and  was  used  by 
this  observer  in  conjunction  with  Wiener  (170)  to  investigate  the  enzyme 
of  the  liver  and  kidney  which  destroys  uric  acid.  It  is  particularly 
valuable  for  animal  tissues  and  consists  essentially  in  first  reducing  the 
tissue  to  a  pulp  by  chopping  finely  and  then  pressing  through  a  fine  sieve 
to  remove  connective  tissue,  etc.  The  mass  is  then  spread  in  thin  layers 
on  glass  plates  and  dried  as  quickly  as  possible  in  a  current  of  warmed 
and  dried  air.  The  dry  film  is  then  scraped  of?  and  ground  to  an  ex- 
tremely fine  powder  in  toluene  in  a  paint  mill.  The  suspension  is 
filtered  by  reduced  pressure  and  washed  with  toluene  to  remove  lipoid 
substances.  On  evaporation  of  the  toluene,  a  fine  dry  powder  is  left, 
which  can  be  kept.  To  obtain  solutions  of  the  enzyme  this  powder  is 
extracted  with  water,  weak  acid  or  alkali,  according  to  the  properties  of 
the  enzyme  in  question,  as  regards  its  solubility,  etc. 

Having  by  one  or  other  of  the  methods  described  obtained  a  solution, 
which  contains  many  other  things  in  addition  to  the  enzyme  desired, 
the  next  step  is  to  purify  the  latter  as  far  as  possible  from  these  foreign 
substances.  Inorganic  salts  and  other  diffusible  bodies  can  be  removed 
to  a  large  extent  by  dialysis,  as  was  done  in  the  case  of  all  three  of  the 
preparations  of  Osborne,  of  Pekelharing  and  of  Fraenkel  and  Hamburg 
mentioned  above.  It  sometimes  happens  that  the  solution  loses  its 
activity  to  a  greater  or  less  degree  during  this  operation.  This  is,  in 
some  cases,  ascribed  to  diffusion  through  the  parchment-paper  and  also 
to  destruction  of  enzyme.  There  is,  however,  another  possible  cause, 
viz.,  the  removal  of  a  co-enzyme,  as  we  shall  see  in  the  eighth  chapter, 
so  that  it  is  necessary  to  examine  the  diffusate  before  drawing  conclu- 
sions. 

To  remove  proteins  and  certain  carbohydrates,  a  very  ingenious 
method  was  used  by  Effront  (54,  p.  126)  and  improved  upon  by  Fraenkel 
and  Hamburg  in  their  work  on  amylase.  This  may  be  called  the 
"biological"  method.  As  stated  above,  it  was  shown  by  Beijerinck 


METHODS  OF  PREPARATION  AND  INVESTIGATION    23 

that  amylase  is  not  a  food-stuff  for  yeast,  so  that  if  solutions  containing 
carbohydrate  and  protein  are  fermented  with  yeast,  these  bodies  will  be 
used  up  and  the  enzyme  will  be  left  behind.  In  the  experiments  of 
Fraenkel  and  Hamburg  the  yeast  was  made  nitrogen-hungry  by  cul- 
tivating it  in  a  nitrogen-poor  medium.  The  method  is  certainly  worth 
trying  with  other  enzymes,  especially  with  the  proteoclastic  ones,  since, 
if  these  are  proteins  in  their  nature,  they  should  serve  as  a  source  of 
nitrogen  to  micro-organisms. 

— *  Having  purified  an  enzyme  solution,  by  dialysis  or  otherwise,  the 
enzyme  is  frequently  precipitated  by  some  means  or  other.  Whatever 
the  precipitant  may  be,  prolonged  contact  with  it  is  deleterious  to  the 
enzyme  to  a  greater  or  less  extent.  Precipitation  by  acetone  or  alcohol 
and  ether  seems  to  be  the  most  innocuous  method,  but  in  some  cases, 
as  in  that  of  zymase  and  other  intracellular  enzymes,  the  precipitate 
must  be  filtered  off  from  the  alcohol  as  quickly  as  possible. 
—Saturation  with  ammonium  sulphate  is  another  useful  method. 

The  carrying  down  of  enzymes  in  adsorbed  form  with  amorphous 
precipitates  is  an  old  method  and  sometimes  is  very  serviceable.  In 
order  that  this  method  may  be  an  effective  one,  it  follows,  from  what 
has  been  said  in  connection  with  colloidal  adsorption-compounds,  that 
the  body  to  be  precipitated  and  the  precipitant  should  be  electrically 
charged  and  also  of  opposite  sign,  unless  an  effective  amount  of  electrolyte 
is  present.  Since  most  solid  particles  in  water  are  electro-negative,  the 
enzyme  solution  should  be  first  made  acid  to  render  it  electro-positive. 
Accordingly,  we  find  that  Michaelis  and  Rona  (116,  117  and  118),  in 
their  method  of  freeing  liquids  from  proteins  or  of  precipitation  of  enzymes 
by  addition  of  mastic  emulsion,  first  slightly  acidulate  the  liquid,  and 
some  enzymes,  e.g.,  trypsin,  require  an  electrolyte  in  the  form  of  a  salt 
for  their  precipitation.  Jacoby's  method  of  producing  a  precipitate  of 
uranyl  phosphate  in  an  enzyme  solution,  which  is  on  occasions  a  valuable 
one,  comes  under  this  head  (92,  pp.  138  and  139). 

If  it  be  desired  to  evaporate  to  dryness  a  solution  of  an  enzyme,  this 
must  be  carried  out  under  reduced  pressure  and  at  as  low  a  temperature 
as  possible.  For  concentrating  such  a  solution  the  apparatus  of  Schulze 
and  Tollens l  will  be  found  useful.  This  consists  in  allowing  the  solution 
to  trickle  along  an  evacuated  glass  spiral,  kept  at  40°  or  lower.  In  this 
way  the  enzyme  is  only  exposed  to  the  raised  temperature  for  a  short 
time;  a  very  effective  evaporation  is  produced,  as  I  can  testify  from 
experience. 

In  the  investigation  of  the  action  of  enzymes  there  are  two  different 
objects  to  be  attained  ;  the  main  problem  may  be  the  chemical  nature 
of  the  bodies  that  are  formed,  or  it  may  be  desired  to  study  the  rate  of 
change  and  the  various  conditions  which  affect  it.  In  the  former  case 
appropriate  chemical  methods  are,  of  course,  made  use  of,  differing  in 
each  case.  Although  it  is  not  the  purpose  of  this  short  monograph  to 
give  details  of  the  numerous  chemical  methods  available,  it  is  advisable 
to  refer  briefly  to  the  method  recently  introduced  by  Sorensen  (174)  on 
account  of  its  theoretical  interest.  This  method  is  base'd  on  the  dis- 
covery of  Schiff  that,  by  the  action  of  formaldehyde  on  amino-acids,  the 
NH2  group  is  neutralised  by  conversion  into  NCH2.  Formaldehyde  is 

*See  Lassar-Cohn,  Arbeits  Mcthoden  M.S.W.,  4te  Aufl;,  Allgem.  Teil,  p.  92,   1906. 


24  THE  NATURE  OF  ENZYME-ACTION 

added  in  excess  to  aproteolytic  digest,  and  the  concentration  of  carboxyl 
groups  can  then  be  estimated  in  the  usual  way  by  titration  with  standard 
alkali.  The  increase  in  amino-acids  and  simple  polypeptides  during 
the  course  of  the  hydrolysis  is  determined  by  this  means.  In  the  latter 
case,  since  it  is  of  importance  to  be  able  to  make  a  large  number  of  ob- 
servations in  a  limited  time,  a  physical  method,  such  as  that  of  optical 
activity -,  will  be  chosen  when  such  a  one  is  available.  Whatever  method 
be  selected,  it  should,  naturally,  be  that  one  which  estimates  that  particular 
property  which  is  subject  to  most  change.  For  instance,  in  following  the 
course  of  the  hydrolysis  of  disaccharides,  the  polarimeter  or  the  copper- 
reducing  power  is  indicated.  When  the  changes  are  not  large,  as  in 
the  investigation  of  reversion  effects,  Croft  Hill's  "  optical  factor  "  (89, 
p.  583)  gives  valuable  help ;  this  is  the  ratio  of  the  optical  activity  to 
the  copper-reducing  power,  and  since,  in  the  production  of  a  disaccharide 
from  glucose,  the  former  goes  up  and  the  latter  down,  the  changes  in 
the  optical  factor  are  larger  than  in  either  of  the  components  alone. 
The  method  also  tends  to  correct  errors  of  estimation.  As  regards  the 
determination  of  the  copper-reducing  power  of  sugars,  the  only  really  re- 
liable method  is  that  of  Allihn,  in  its  various  modifications.  The  standard 
method  will  be  found  in  the  work  of  Adrian  Brown  (38,  p.  78)  referred 
to  in  the  list  at  the  end  of  this  book,  while  the  manner  of  preparing  for 
sugar-estimation  solutions  containing  enzymes  is  given  in  the  paper  by 
Aders  Plimmer  (133,  p.  23). 

The  measurement  of  optical  activity  is  also  useful  in  the  case  of  some 
proteoclastic  enzymes,  especially  when  acting  on  pure  polypeptides ;  it 
has  been  used  for  this  purpose  by  Fischer  and  Abderhalden  (65,  p.  57) 
and  by  Euler  (58).  It  is  also  available  in  the  case  of  emulsin  acting  on 
glucosides. 

Changes  of  viscosity  are  very  marked  in  the  action  of  proteoclastic 
enzymes  on  most  proteins,  which  are,  as  a  rule,  in  their  unaltered  con- 
dition, bodies  with  considerable  viscosity  (Spriggs  [146]).  The  method 
has  its  limitations,  since  it  gives  but  little  information  as  to  the  essential 
chemical  work  of  the  enzyme.  This  may  be  seen  by  comparing  the 
change  of  viscosity  with  the  nitrogen-content  of  the  filtrate  after  pre- 
cipitation by  tannin,  in  the  case  of  trypsin.  It  is  found  that  for  some 
time  after  the  change  in  viscosity  has  come  to  a  standstill,  the  production 
of  peptone  and  amino-acids  continues  at  a  considerable  rate  (13).  In 
other  words,  the  reaction  estimated  by  change  of  viscosity  would  appear 
to  have  come  to  an  end,  while  it  is  actually  proceeding  at  a  fair  rate. 
The  same  thing  may  be  said  about  other  similar  changes  in  physical 
consistency,  such  as  are  at  the  basis  of  the  methods  of  Griitzner  (70)  and 
of  Vernon  (161,  p.  406)  with  liquefaction  of  fibrin,  of  that  of  Fermi  with 
liquefaction  of  gelatin  and  the  various  forms  of  Mett's  tubes.  After 
the  fibrin  flakes  have  disappeared,  the  protein  will  be  still  found  to  exist 
in  a  state  which  coagulates  on  boiling.  As  to  gelatin,  its  capacity  of 
setting  when  cooled  is  destroyed  with  extreme  rapidity  by  trypsin.  I 
found  that  five  or  six  minutes'  action  of  the  enzyme  at  40°  was  enough 
for  the  purpose,  whereas  very  little  chemical  change  had  taken  place  in 
this  time,  as  shown  by  the  small  amount  of  the  change  in  electrical 
conductivity.  The  chief  use  of  the  above  methods  is  to  indicate  the 
presence  of  a  proteoclastic  enzyme,  or  to  compare  the  relative  concentra- 
tions of  two  solutions  of  the  same  enzyme. 


METHODS  OF  PREPARATION  AND  INVESTIGATION    25 

When  changes  of  volume  occur  in  the  course  of  the  reaction,  dilato- 
metric  measurements  may  be  made. 

Measurements  of  changes  in  molecular  state  would,  in  many  cases, 
afford  valuable  indications  of  the  work  of  the  enzyme.  Such  may  be 
obtained  {VQ\^.  freezing-point  (12^}  or  perhaps  vapour-pressure  determina- 
tions ;  but,  so  far  as  I  am  aware,  no  research  of  this  nature  has  yet  been 
undertaken. 

To  return  to  optical  methods,  the  refractive  index  may  be  used, 
but,  as  a  rule,  its  changes  are  small  in  the  reactions  which  concern  us 
here. 

On  the  other  hand,  the  spectre-photometer  or  colorimeter  has  been 
made  frequent  use  of.  The  oxidation  of  the  leuco-base  of  malachite- 
green  to  the  green  pigment  itself  under  the  action  of  peroxidases,  as 
investigated  by  Czyblarz  and  v.  Fiirth  (173),  is  a  favourable  case  for  such 
a  method.  The  course  of  the  change  indicated  by  the  biuret  reaction 
has  been  followed,  by  Klug  (99,  p.  43)  and  by  myself  (13,  pp.  279  and 
289),  with  the  aid  of  the  spectro-photometer.  In  using  the  method  for 
colour  tests  certain  difficulties  have  yet  to  be  overcome,  with  regard  to 
which  the  original  papers  must  be  consulted.  It  is  possible  that 
it  may  be  made  available  for  Millon's  and  the  tryptophane  colour 
reactions. 

The  last  method  to  be  referred  to  here  is  that  of  changes  in 
electrical  conductivity.  Sjoqvist  (145)  found  that  protein  solutions 
showed  a  diminution  of  this  property  under  the  action  of  pepsin.  The 
fact  was  confirmed  by  Oker-Blom  (125),  who  also  showed  that  the 
action  of  trypsin  was  accompanied  by  a  rise  in  conductivity  and  advo- 
cated the  method  as  of  great  convenience.  It  has,  notwithstanding, 
been  hitherto  used  but  little  in  the  investigation  of  enzymes.  Victor 
Henri  and  Larguier  des  Bancels  (82)  have  made  a  few  observations 
with  respect  to  the  action  of  trypsin  on  gelatin,  and  I  have  myself  made 
a  somewhat  extensive  use  of  it  for  the  general  action  of  the  same 
enzyme  (13  and  16).  It  showed  itself  to  be  of  special  value  in  this  in- 
stance, since  it  was  found  to  follow  exactly  the  same  time-course  as  the 
nitrogen  in  the  tannic  filtrate,  the  cause  of  the  rise  being  almost  entirely 
due  to  the  production  of  peptones  and  amino-acids  (16).  The  method 
may  also  be  used  for  other  proteoclastic  enzymes  acting  in  alkaline  or 
neutral  solutions.  The  change  is  more  complex  in  acid  solutions  since 
the  products  of  the  reaction  have  a  greater  affinity  for  acid  than  the 
original  substrate  has ;  there  is  therefore  at  the  beginning  a  fall  of  con- 
ductivity owing  to  diminution  of  hydrion.  This  subsequently  gives 
place  to  a  rise,  but  the  exact  course  of  the  change  hasi  not  yet  been 
worked  out. 

The  action  of  lipase  on  esters  of  the  lower  fatty  acids  may  be  fol- 
lowed in  this  manner,  since  there  is  a  considerable  rise  in  conductivity 
owing  to  the  formation  of  free  acid. 

It  is  naturally  not  a  good  method  for  enzymes  acting  on  carbohy- 
drates, since  there  is,  as  a  rule,  no  production  of  electrolytes  in  these 
reactions.  An  exception  is  the  hydrolysis  of  sinigrin  by  myrosin, 
in  which  a  good  conductor,  vtz.,  potassium  hydrogen  sulphate,  is  pro- 
duced. 

In  the  measurement  of  conductivity,  the  usual  method  of  Kohl- 
rausch  may  be  used,  but  it  is  better  to  use  Whetham's  modification 


26  THE  NATURE  OF  ENZYME-ACTION 

with  rotating  commutator  (168,  p.  331),  since  there  is  no  necessity  in 
this  method  to  use  electrodes  coated  with  platinum-black,  which  has 
a  certain  decomposing  effect  on  many  substances.  The  method  has 
also  the  convenience  that  ordinary  batteries  and  galvanometer  are 
used. 

When  comparing  the  action  of  different  strengths  of  enzyme  solu- 
tions, it  is  advisable  to  take  as  the  basis  of  comparison  the  times  taken 
to  effect  an  equal  change,  rather  than  the  amounts  of  change  in  equal 
times.  This  is  especially  important  where  the  reaction  takes  place  in 
stages,  since  only  in  this  manner  is  it  possible  to  have  comparable 
values.  Instances  of  such  reactions  are  the  action  of  amylase  on  starch 
and  proteoclastic  actions  in  general.  If  the  reaction  has  not  arrived  at 
the  same  stage  in  the  cases  to  be  compared,  there  is  the  source  of  error 
due  to  the  various  stages  not  taking  place  with  the  same  velocity. 

In  the  case  of  trypsin,  for  example,  proteoses  are  produced  very 
rapidly  and  are  again  converted  fairly  rapidly  into  peptone,  while  this 
latter  body  is,  in  part,  hydrolysed  further  but  more  slowly  into  amino- 
acids ;  at  the  same  time  some  amino-acids  are  produced  from  the  first, 
so  that  the  reaction  is  a  very  complicated  one,  and  the  results  obtained 
at  different  stages  would  be  very  difficult  to  interpret,  unless  times  of 
equal  change  are  chosen  for  comparison. 

Another  practical  question  may  be  touched  upon  to  conclude  this 
chapter.  When  the  reaction  has  to  be  stopped  at  a  given  stage  for  the 
purpose  of  determining  how  far  it  has  gone,  as  is  necessary  in  the  use 
of  most  of  the  methods  described,  with  the  exception  of  that  of  electri- 
cal conductivity  or,  in  some  cases,  the  optical  activity,  one  must  be  able 
to  stop  the  action  of  the  enzyme  by  some  means.  Now  it  is  by  no 
means  a  matter  of  indifference  how  this  is  done.  As  will  be  shown  in 
detail  in  a  later  chapter,  the  activity  of  enzymes  is  enormously  in- 
creased by  rise  of  temperature,  although  in  the  end  abolished.  The 
method  of  taking  a  certain  volume  of  the  reacting  mixture  and  heating 
this  to  boiling  point,  or  in  a  steam  steriliser,  as  is  frequently  done,  is 
liable  to  be,  and  in  fact  has  been,  the  source  of  errors.  It  is  plain  that 
the  solution  cannot  be  heated  instantaneously  to  the  destruction 
temperature  of  the  enzyme,  and,  during  the  time  taken  for  this  to  be 
attained,  the  activity  of  the  enzyme  is  enormously  increased.  In  this 
way  changes  take  place  after  the  action  is  supposed  to  have  ceased. 
If  it  is  requisite  that  the  action  should  be  stopped  by  heat,  the  solution 
may  be  run  in  a  thin  stream  into  boiling  water.  If  not  desirable  to 
dilute  the  mixture,  it  may  be  cooled  to  o°,  or  better,  frozen  solid  and 
kept  so  until  wanted  for  investigation.  When  the  addition  of  chemical 
reagents  is  immaterial,  this  means  of  stopping  the  action  of  an  enzyme 
is  most  convenient.  Ammonia  may  be  added  to  invertase  experi- 
ments ;  in  this  case  it  is  of  use  in  another  way,  in  that  it  gets  rid  of  the 
bi-rotation  of  glucose,  by  bringing  the  glucose  system  to  an  equilibrium 
at  once.  Alkali  may  be  added  to  pepsin  or  acid  to  trypsin.  Of  course 
when  a  precipitant,  such  as  tannin  or  phospho-tungstic  acid,  is  used  in 
the  further  stages  of  the  method  it  may  be  added  at  once  to  stop  the 
action. 

It  may  be  noted  that  the  advantage  of  the  electrical  conductivity 
method  is  that  readings  can  be  taken,  by  using  appropriate  vessels, 
without  disturbing  the  course  of  the  reaction.  The  same  thing  applies 


METHODS  OF  PREPARATION  AND  INVESTIGATION    27 

to  the  viscosity  method  and  to  the  polarimeter  method,  if  a  jacketed 
tube  be  used  to  keep  the  solution  at  a  constant  temperature. 

Nothing  has  been  said  as  to  the  maintenance  of  a  constant  tempera- 
ture in  following  the  course  of  enzyme-action,  important  as  it  is.  The 
best  means  of  doing  this  can  easily  be  found  by  consulting  such  a  work 
as  that  of  Ostwald  and  Luther  on  physico-chemical  measurements. 


CHAPTER  V. 

REVERSIBILITY  OF  ENZYME-ACTION. 

REFERENCE  has  already  been  made  in  the  first  chapter  to  the  fact  that, 
in  the  case  of  such  a  reaction  as  that  resulting  in  the  equilibrium 
between  methyl  acetate,  acetic  acid,  methyl  alcohol  and  water,  both 
the  hydrolysis  of  the  ester  and  also  its  formation  from  the  acid  and 
alcohol  are  accelerated  by  a  catalyst  such  as  hydrochloric  acid.  If 
therefore  enzymes  are  to  fall  into  line  with  other  catalysts,  they  will 
also  accelerate  synthetic  processes. 

We  know  .that  many  processes  of  the  kind  known  as  reversible,  or 
balanced,  reactions  take  place  in  the  living  organism.  Particularly 
obvious  are  those  cases  where  material  is  stored  up  in  an  insoluble  form, 
like  starch  or  glycogen.  These  bodies  are,  under  certain  conditions,  syn- 
thesised  from  sugars  and,  under  other  conditions,  are  hydrolysed  back 
again,  when  required. 

Since  the  first  definite  proof  of  a  synthetic  process  taking  place 
under  the  influence  of  an  enzyme  was  brought  forward  by  Croft  Hiil 
(86),  so  many  other  cases  have  been  discovered  that  it  is  no  longer 
necessary  to  give  a  list  of  them.  In  fact  the  impression  is  distinctly 
given  that  it  is  merely  a  question  of  finding  the  proper  conditions  in 
order  to  be  able  to  obtain  synthesis  from  all  enzymes. 

As  regards  these  conditions  the  first  thing  that  requires  attention  is 
the  part  played  by  water.  Methyl  acetate  can  be  kept  indefinitely  in  a 
closed  bottle  without  change,  but  the  presence  of  the  smallest  amount 
of  water  causes  the  hydrolysis  of  a  part  of  it,  and  the  greater  the  propor- 
tion of  water,  the  more  the  reaction  takes  place  in  the  direction  of  the 
formation  of  acetic  acid  and  alcohol.  Stated  in  another  way,  the 
greater  the  relative  concentration  of  the  water  component,  the  nearer 
the  equilibrium-point  is  to  the  position  of  complete  hydrolysis. 

Amongst  enzymes  there  are  many  instances  where  the  hydrolysis 
appears  to  be  complete,  invertase  for  example.  But  it  has  been  shown 
by  Visser  (164,  p.  275)  that  a  O'25N  solution  of  saccharose  gave  only  a 
rotation  of-  3*26°  when  acted  on  by  invertase  until  no  further  change 
took  place,  whereas  when  inverted  by  acid  the  final  rotation  was-  3*42°, 
which  is  what  the  reading  should  be  if  the  solution  contained  only 
glucose  and  fructose.  And  again,  a  particular  solution  containing  equal 
amounts  of  glucose  and  fructose  had  an  initial  rotation  of  -i2'46°; 
after  the  action  of  invertase  for  two  months  the  rotation  had  fallen  to 
-  12*29°.  A  change  of  this  degree  means  that  an  equilibrium  position 
exists  when  about  99  per  cent,  of  the  saccharose  is  hydrolysed,  and 
that,  if  the  products,  glucose  and  fructose,  be  exposed  to  the  enzyme, 
a  formation  of  saccharose  to  this  extent  takes  place.  When  we  remem- 
ber that  the  equilibrium  position  is  given  by  the  ratio  of  the  velocity  of 

28 


REVERSIBILITY  OF  ENZYME-ACTION  29 

the  hydrolytic  to  that  of  the  synthetic  process,  we  see  at  once  how  very 
much  the  former  exceeds  the  latter.  Visser  in  fact  found  that,  for  O'5N 
saccharose,  the  equilibrium-constant,  i.e.,  the  ratio  of  the  two  velocity- 
constants,  was  very  nearly  50,  so  that,  since  six  days  are  required  to 
attain  equilibrium  when  saccharose  of  this  concentration  is  acted  upon 
by  invertase,  about  ten  months  (/>.,  fifty  times  six  days)  would  be 
needed  for  the  reverse  reaction  (164,  p.  301). 

Now  it  might  be  thought  that  a  synthesis  of  so  small  a  degree  could 
not  be  of  much  practical  importance.  This  would  be  an  error,  as  the 
following  considerations  will  show.  Let  us  take  the  case  of  amylase, 
where  a  similar  reaction,  progressing  almost  to  completion,  occurs,  and 
let  us  suppose  that  no  more  than  I  per  cent,  of  starch  is  formed  when 
the  enzyme  acts  upon  maltose  or  dextrin.  Since  the  product  is  an  in- 
soluble body  the  equilibrium  will  exist  only  for  a  moment,  so  that  more 
starch  will  be  formed ;  as  the  rate  of  this  reaction  is  slow,  as  shown 
above,  the  amount  of  starch  formed  per  unit  time  will  not  be  great, 
although  by  no  means  negligible.  The  process,  it  will  be  noted,  is 
analogous  to  that  of  the  precipitation  of  chloride  as  silver  salt.  It  is 
most  likely,  as  Croft  Hill  points  out,  that  the  storage  of  starch  in  the 
plant  and  that  of  glycogen  in  the  animal  are  to  be  explained  on  these 
lines  (86  and  88). 

Moreover,  it  is  not  necessary,  in  order  that  considerable  synthesis 
may  take  place  when  the  equilibrium-point  is  close  to  that  of  complete 
hydrolysis,  that  the  synthetic  product  should  be  deposited  in  an  insolu- 
ble form ;  it  may  be  removed  from  the  reacting  system  by  any  other 
means,  such  as  diffusion,  into  blood-current  or  elsewhere,  or  taken  up  in 
some  other  independent  reaction. 

The  simplest  case  of  reversibility  is  that  of  lipase  acting  on  esters  of 
lower  fatty  acids,  which  was  first  investigated  by  Kastle  and  Loevenhart 
(95);  we  will  therefore  briefly  examine  this  reaction.  It  may  perhaps 
seem  strange  that  the  action  of  maltase  on  glucose,  in  which  the  syn- 
thetic action  of  enzymes  was  first  discovered  by  Croft  Hill,  has  not  yet 
been  dealt  with.  The  reason  for  this  omission  is  that  the  conditions 
here  have  turned  out  to  be  complicated  by  the  existence  of  the  two 
optical  isomers  of  the  bi-hexose,  which  is  formed,  so  that  the  reaction 
will  best  be  discussed  at  a  later  stage. 

It  is  quite  easy  to  observe  the  production  of  ethyl  butyrate  when 
lipase  acts  on  a  mixture  of  ethyl  alcohol  and  butyric  acid,  since  the 
ester  has  a  characteristic  odour,  very  different  from  the  acid  or  alcohol. 
To  quote  the  authors  named  :  "  When  a  fresh  aqueous  extract  of  pancreas 
is  treated  with  a  mixture  of  dilute  butyric  acid  (0*1  to  o*O5N)  and  ethyl 
alcohol  (sufficient  to  bring  the  whole  to  1*5  per  cent.),  the  very  character- 
istic odour  of  ethyl  butyrate  soon  develops  even  at  the  ordinary  temper- 
ature and  in  the  presence  of  antiseptics,  whereas  if  the  pancreatic  ex- 
tract is  first  boiled  the  mixture  never  develops  the  odour  of  the  ester". 
If  the  experiment  be  done  on  a  large  scale,  the  ester  can  be  separated  by 
distillation  and  can  be  hydrolysed  back  again  by  the  same  enzyme  that 
produced  it.  Moreover,  when  ethyl  butyrate  is  hydrolysed  by  lipase, 
the  reaction  is  never  complete,  so  that,  in  other  words,  an  equilibrium 
is  arrived  at.  It  is  of  interest  that  the  hydrolysis  does  not  proceed  so 
far  when  effected  by  lipase  as  when  effected  by  hydrochloric  acid,  as 
has  been  recently  shown  by  Dietz  (48,  p.  320),  As  was  pointed  out  in 


30  THE  NATURE  OF  ENZYME-ACTION 

the  first  chapter  of  this  book,  this  circumstance  does  not  mean  that  the 
enzyme  does  not  follow  the  general  laws  of  catalysis,  but  that  its  mode 
of  action  is  by  the  formation  of  some  kind  of  compound  between  enzyme 
and  ester  on  the  one  side  and  between  enzyme  and  products  on  the 
other  side. 

Now  pancreatic  lipase  hydrolyses  the  higher  fats  as  well  as  the 
simpler  esters ;  synthetic  production  of  higher  esters  of  glycerol  would 
therefore  be  expected  to  take  place  and  has  been  actually  observed ;  in 
fact,  Hanriot  (72)  has  obtained  by  means  of  lipase  a  butyric  ester  of 
glycerol,  monobutyrin,  and  Pottevin  has  obtained  mono-  and  tri-olein 

(135). 

The  physiological  importance  of  this  reversibility  of  lipase-action  is 
pointed  out  by  Loevenhart  (in)  himself.  In  the  process  of  digestion 
and  absorption  of  fat  there  is  no  doubt  that  fat  globules  are  found  in 
the  cells  of  the  intestinal  mucous  membrane  and  that  fat  taken  as  food 
is  hydrolysed  in  the  lumen  of  the  intestine.  There  must  therefore  be 
some  mechanism  by  which  the  products  of  hydrolysis  are  resynthesized 
in  the  cells  after  absorption.  It  is  obvious  that  if  a  Jipase  were  present 
in  these  cells,  it  would  be  capable  of  considerable  synthetic  action,  since 
the  fat  produced,  being  insoluble,  is  deposited  out  of  the  reacting 
system  in  the  form  of  droplets.  Loevenhart  has  in  point  of  fact  been 
able  to  obtain  a  lipase  from  intestinal  mucous  membrane  of  the  pig 
after  thoroughly  washing  away  the  pancreatic  enzymes.  A  similar 
enzyme  was  also  obtained  from  the  liver  and  other  places  where  fat 
storage  occurs.  In  all  these  cases,  when  the  blood  and  lymph  bathing 
the  cells  becomes  poor  in  fatty  acid  and  glycerol,  either  owing  to  fat 
being  stored  elsewhere  or  to  its  being  used  up  by  oxidation,  as  in 
starvation,  the  lipase  restores  equilibrium  by  effecting  hydrolysis  of  the 
fat  which  had  previously  been  stored  up. 

Before  proceeding  to  the  more  difficult  cases  of  synthetic  action  on 
carbohydrates,  it  is  necessary  to  say  a  few  words  about  the  stereo- 
chemistry of  the  glucosides.  These  bodies  are  recognised  as  having 
the  internal  anhydride  structure  of  a  7-lactone.  Glucose  itself  has  the 
same  structure  and  is  therefore  capable  of  existence  in  two  optically 
isomeric  forms,  known  as  a-  and  /3-glucose,  which  differ  from  one 
another  in  the  relative  positions  of  the  H  and  OH  of  the  terminal 
aldehyde  group  as  shown  in  the  diagrams : — 

OH   H  H    OH 

\/  \x 

C COH  C COH 


II 


OH 
H      \ 


H 


OH 
H      \ 


C C— H  C C— H 

HCOH  'HCOH 

HCOH  HCOH 

H  H 

o-glucose.  /8-glucose. 

One  of  these  two  forms,  which  we  call,  for  convenience,  the  a-form, 
has  a  greater  specific  rotation  than  the  /3-form.     In  glucose  in  the  dry 


REVERSIBILITY  OF  ENZYME-ACTION  31 

state  the  first  preponderates ;  when  dissolved  in  water  the  rotation  is  at 
first  higher  than  after  standing,  and  this  decreases  until  a  state  of  equi- 
librium is  finally  established.  According  to  Tanret,  in  a  10  per  cent, 
solution  in  equilibrium  there  exists  37  per  cent,  of  the  a-form  and  6*3 
per  cent,  of  the  /3-form.  It  was  found  by  Emil  Fischer  (64)  that,  when 
a  solution  of  glucose  in  methyl  alcohol,  in  which  presumably  both  forms 
of  the  sugar  are  present,  is  acted  on  by  hydrochloric  acid,  two  methyl- 
glucosides  are  formed.  One  of  these,  the  a-form,  corresponds  to  the 
left-hand  formula  of  the  two  above,  in  which  the  hydrogen  of  the 
uppermost  OH  is  replaced  by  CH3,  while  the  /3-methylglucoside  is 
similarly  formed  from  the  other. 

In  all  yeasts  which  are  able  to  ferment  maltose,  as  shown  by 
Fischer,  there  is  present  an  enzyme  which  is  able  to  hydrolyse  the  mal- 
tose as  a  necessary  preliminary  to  the  action  of  zymase.  This  enzyme 
is  known  as  maltase  and  is  not  identical  with  invertase.  Now  it  is 
found  that  maltase  will  hydrolyse  the  a-methylglucoside,  but  not  the 
/3-form,  whereas  emulsin,  the  enzyme  found  in  bitter-almonds  and  else- 
where, which  acts  upon  most  of  the  natural  glucosides,  such  as  amyg- 
dalin,  salicin,  etc.,  will  not  touch  the  a-glucoside,  but  readily  hydrolyses 
the  /3-form.  The  conclusion  is  that  maltose  has  the  structure  of  an  a- 
glucoside  and  that  the  natural  glucosides,  such  as  salicin,  are  /3-glucosides. 

If  maltase  be  allowed  to  act  upon  a  strong  solution  of  glucose  we 
should  expect  that,  since  this  enzyme  hydrolyses  maltose  to  glucose,  if 
any  synthetic  process  takes  place,  maltose  would  be  formed,  and  this 
was  in  fact  what  Croft  Hill  (86)  at  first  believed  to  take  place.  It 
turned  out,  however,  on  separating  the  bi-hexose  which  was  produced, 
that  it  consisted  only  partially  of  maltose,  the  rest  being,  as  supposed, 
a  new  sugar,  revertose  (89).  Emmerling  (56)  showed  later  that  this 
"revertose"  was  actually  isomaltose,  and  Frankland  Armstrong  (5,  vii., 
p.  598)  has  confirmed  this.  What  then  is  isomaltose?  It  is  a  bi- 
hexose  which  is  hydrolysed  by  emulsin  with  the  production  of  two 
molecules  of  glucose,  so  that  we  are  justified  in  regarding  it  as  the 
optical  isomer  of  maltose,  viz.,  the  y8-glucose-glucoside.  Now  accord- 
ing to  the  hypothesis  first  put  forward  by  van't  Hoff  (90,  p.  12)  and 
accepted  by  Croft  Hill,  an  enzyme  only  synthesizes  the  same  body 
which  under  other  conditions  it  hydrolyses.  Moreover,  this  is  the  only 
possible  hypothesis  if  enzymes  are  to  be  brought  into  line  with  other 
catalysts.  It  is  therefore  of  some  importance  to  examine  more  closely 
the  facts  as  far  as  we  know  them.  Isomaltose  is  not  hydrolysed  by 
maltase,  but  by  emulsin ;  it  is  therefore,  to  begin  with,  rather  startling 
and  suggestive  that,  in  Croft  Hill's  experiments,  the  synthetic  products 
are  almost  completely  hydrolysed  back  by  the  same  enzyme  prepara- 
tion that  produced  them.  For  example,  a  45  per  cent,  solution  of 
glucose  was  acted  on  by  yeast  extract  until  its  "optical  factor"  (see 
earlier,  Chapter  IV.)  had  been  raised  from  0*525  to  0-676,  which  indi- 
cates a  synthesis  of  15  per  cent,  reckoned  as  maltose.  After  boiling, 
10  c.c.  of  this  solution  was  diluted  to  200  c.c.,  some  of  the  original 
yeast  extract  was  added  and  it  was  allowed  to  stand  at  28°  tor  ten  days. 
The  optical  factor  was  now  reduced  to  0*537,  indicating  almost  com- 
plete hydrolysis,  while  a  control  with  the  same  amount  of  boiled  en- 
zyme solution  showed  only  a  reduction  to  0*675  (89,  p.  585).  Further, 
it  was  found  that  if  the  synthetical  products  were  fermented  by  a  pure 


32  THE  NATURE  OF  ENZYME-ACTION 

maltase-containing  yeast,  such  as  Saccharomyces  Ellipsoideus  /.,  only  a 
part  of  the  bi-hexose  was  hydrolysed  and  fermented,  presumably  the 
maltose  alone.  It  seems  to  me  that  the  explanation  of  the  production 
of  isomaltose  lies  in  these  facts.  In  the  first  place  it  is  to  be  noted 
that  the  enzyme  solutions  used  were  made  from  ordinary  brewer's 
yeast,  and  that  these  extracts  hydrolysed  practically  the  whole  of  the 
synthetical  products,  whereas  pure  maltase  only  acted  on  a  part.  Croft 
Hill  himself  suggested  the  presence  of  a  mixture  of  enzymes  in  his  ex- 
tracts, and  since  that  time  it  has  been  shown  that  many  yeasts  contain 
emulsin  (Henry  and  Auld  [85]) ;  in  fact  ordinary  pressed-yeast  will 
rapidly  hydrolyse  amygdalin  at  a  temperature  of  38°,  as  can  easily  be 
verified.  The  conclusion  to  be  drawn  is  that  the  formation  of  isomal- 
tose can  be  satisfactorily  explained  as  being  due  to  emulsin  and  that  of 
maltose  by  the  presence  of  maltase. 

Again,  Fischer  and  Armstrong  (188)  found  that,  by  the  action  of 
Kefir-lactase  on  a  mixture  of  glucose  and  galactose,  isolactose  and  riot 
lactose  was  formed.  On  referring  to  the  table  given  on  p.  315!  °f 
their  paper,  the  significant  fact  will  be  noticed  that  this  synthetic  pro- 
duct was  hydrolysed,  in  dilute  solution,  by  the  same  enzyme,  or 
mixture  of  enzymes  (Kefir-lactase),  that  produced  it  under  other  condi- 
tions. It  is  clear,  therefore,  that  the  enzyme  preparation  contained  a 
body  capable  of  hydrolysing  isolactose,  and  there  is  no  reason  to  sup- 
pose that  the  synthesis  was  effected  under  the  influence  of  any  other 
enzyme. 

A  quite  different  point  of  view  has  been  taken  by  Frankland  Arm- 
strong (5,  vii.),  who  regards  it  as  the  rule  that  an  enzyme  synthesizes 
exactly  those  bodies  which  it  does  not  hydrolyse.  Now  it  seems  to  me 
that  such  a  view  is  calculated  only  to  throw  the  whole  subject  into  con- 
fusion ;  but,  apart  from  this,  there  are  many  reasons  for  not  accepting 
it.  It  was  shown  by  Croft  Hill  that  in  the  action  of  maltase  on  glucose 
an  equilibrium-point  was  reached,  in  the  conditions  under  which  he 
worked,  when  about  1 5  per  cent,  of  the  glucose  had  been  synthesized 
to  the  bi-hexose  and  that  the  reaction  then  ceased.  If  the  synthetic 
body  were  incapable  of  hydrolysis  by  the  enzymes  present,  there  is  no 
conceivable  reason  why  the  reaction  should  cease  until  the  whole  of  the 
glucose  is  converted,  because,  as  it  is  not  acted  upon,  it  is  withdrawn 
from  the  sphere  of  action  as  soon  as  it  is  formed.  Granting  that  the 
body  isolated  by  Frankland  Armstrong  from  the  action  of  yeast  extract 
on  glucose  was  isomaltose,  it  must  be  remembered  that  the  yeast  used 
was  ordinary  brewer's  yeast,  which  was  shown  by  Henry  and  Auld  to 
contain  emulsin. 

This  explanation  of  the  results  of  experiments  on  synthesis  of  bi- 
hexoses  may  be  objected  to  for  the  reason  that  the  amount  of  maltose 
found  by  Croft  Hill  was  much  less  than  that  of  "  revertose  "  or  isomal- 
tose. The  cause  of  this  fact  is  no  doubt  that  given  by  the  experimenter 
himself,  viz.,  that  the  yeast  used  contained  amylase  or  dextrinase,  under 
the  synthetic  action  of  which  the  maltose  was  partially  converted  into 
dextrin  ;  bodies  of  this  nature  did  in  fact  make  their  appearance  in  the 
process  of  separating  the  products. 

The  conclusion  we  come  to  is,  then,  that  there  is  no  cogent  evidence 
that  enzymes  produce  by  synthesis  any  bodies  different  from  those 
which  they  hydrolyse. 


REVERSIBILITY  OF  ENZYME-ACTION  33 

The  suggestion  has  been  made  that  synthetic  processes  are  brought 
about  by  special  enzymes,  which  do  not  hydrolyse.  As  yet  no  enzyme 
having  this  property  has  been  prepared.  We  have  seen,  moreover,  that 
the  hypothesis  of  the  existence  of  such  bodies  is  unnecessary  in  the 
present  state  of  our  knowledge  and,  therefore,  according  to  the  canons 
of  scientific  method,  must  be  rejected.  It  is  difficult  also  to  reconcile 
such  a  view  with  the  facts  concerning  the  equilibrium  position.  In  all 
the  experiments  in  which  attention  was  directed  to  this  point,  such  as 
those  of  Croft  Hill,  Visser  and  Dietz,  it  was  found  that  this  equilibrium- 
point  was  the  same  under  the  same  conditions  of  concentration,  etc., 
whatever  the  amount  of  the  enzyme  used,  and  that  different  preparations 
also  gave  the  same  result.  If  the  enzyme  preparation  were  a  mixture 
of  a  hydrolysing  and  a  synthesizing  enzyme,  it  seems  extremely  unlikely 
that  the  two  bodies  should  always  be  present  in  the  same  relative  pro- 
portions. Again,  it  is  found  that  synthesis  takes  place  chiefly  under 
conditions  in  which,  according  to  the  manner  of  reversible  reactions,  it 
would  be  expected  to  occur,  viz.t  high  concentration  of  the  products  of 
hydrolysis. 

A  somewhat  peculiar  position  is  taken  by  Euler  (59  and  60)  on  the 
strength  of  an  experiment  of  Beitzke  and  Neuberg  (23),  who  showed 
that  on  subcutaneous  injection  of  emulsin  into  a  rabbit,  antiemulsin  was 
formed  and  that  this  body  was  able  to  synthesize  a  disaccharide  from 
glucose.  Further  investigation  of  this  rather  surprising  result  is  neces- 
sary before  forming  any  opinion  as  to  its  meaning.  In  any  case,  it 
seems  premature  to  assume,  as  Euler  has  done,  that  antibodies  in  general 
are  the  synthesizing  enzymes  of  the  hypothesis  of  the  previous  para- 
graph. 

Reference  has  already  been  made  to  the  fact  that  the  final  equilibrium 
is  not  the  same  in  the  case  of  enzyme-action  as  in  catalysis  by  acids. 
This  indicates  that  the  enzyme  itself  in  some  way  enters  into  the  com- 
ponents of  that  equilibrium.  Reasons  will  be  given  subsequently  for 
holding  that  there  are  compounds  of  a  certain  kind,  probably  of  the 
nature  of  adsorption,  between  enzyme  and  substrate  and  between  enzyme 
and  products.  As  Euler  points  out,  if  the  formation  and  decomposition 
of  the  former  set  does  not  proceed  at  the  same  rate  as  the  similar  changes 
in  the  latter  set,  it  follows  that  the  enzyme  will  remain  as  a  constituent 
of  the  system  in  equilibrium.  Tammann  had  already  pointed  out  this 
phenomenon  and  showed  that  by  adding  more  enzyme  to  a  system  in 
equilibrium  a  further  change  could  be  caused  to  take  place.  This  result 
was  obtained  in  the  case  of  the  hydrolysis  of  amygdalin  by  emulsin.  I 
have  myself  found  the  statement  to  hold  for  tiypsin.  Similarly,  an  in- 
crease of  hydrolysis  was  obtained  in  a  stationary  system  by  altering  any 
of  the  other  conditions  of  the  equilibrium,  such  as  addition  of  more 
amygdalin,  removal  of  products  of  reaction,  raising  the  temperature  or 
dilution.  It  can  thus  be  seen  that  the  stationary  condition  was  one  of 
true  equilibrium  and  that  to  call  it  a  "  false "  equilibrium  is  incorrect 
(152,  153,  154;  see  also  33,  p.  194,  and  95). 

The  facts  just  referred  to  show  that  the  inactivity  of*  the  enzyme 
was  not  due  to  destruction,  since  it  could  be  made  active  again  by  alter- 
ing the  conditions.  On  the  other  hand  some  enzymes  are  so  unstable 
that  they  become  destroyed  in  the  course  of  the  reaction.  Partial  de- 
struction occurs  even  in  the  case  of  emulsin,  as  shown  by  Tammann 

3 


34  THE  NATURE  OF  ENZYME-ACTION 

( 1 54) ;  and  complete  destruction  of  trypsin  occurs  when  the  action  is 
allowed  to  go  on  for  a  long  time.  There  is  then  both  a  reversible  and 
an  irreversible  state  of  inactivity  of  an  enzyme,  the  former  only  being 
an  equilibrium. 

Nothing  has  yet  been  said  as  to  synthetic  action  on  the  part  of  pro- 
teoclastic  enzymes.  As  pointed  out  by  Leathes  (i  10,  p.  132),  the  facts 
as  to  protein  synthesis  in  the  organism  distinctly  indicate  a  reversible 
enzyme-action.  The  experiments  of  Loewi  (114),  taken  in  conjunction 
with  those  of  Henriques  and  Hansen  (83),  have  shown  that  animals  are 
able  to  maintain  their  nitrogen-content  on  a  diet  in  which  the  only  nitro- 
genous bodies  are  the  products  of  a  prolonged  tryptic  and  ereptic 
digestion  ;  but  that  the  products  of  acid  hydrolysis  are  unable  to  take 
their  place.  To  quote  the  words  of  Leathes :  "  There  appears  to  be  some 
kind  of  linkage  between  certain  groups  in  the  protein  molecules  which 
is  not  uncoupled  by  the  enzymes  in  the  body,  and  that  when  it  is  un- 
coupled, as  in  acid  hydrolysis,  it  is  impossible  for  it  to  be  coupled  up 
again  in  the  body.  This  combination,  which  the  cells  can  neither  take 
to  pieces  nor  put  together  again,  must  be  present,  in  order  that  the  other 
component  parts  of  the  protein  molecule  may  gather  about  it  and  group 
themselves  round  it  when  the  synthesis  of  portein  is  to  occur.  These 
considerations  appear  to  suggest  that  the  synthetic  processes  here  in- 
volved may  be  the  work  of  the  same  agent  as  the  hydrolytic,  the  limita- 
tions in  its  hydrolytic  power  determining  the  limitations  of  its  synthetic 
activity,  as  in  reversible  zymolysis." 

More  direct  evidence  of  protein  synthesis  is  not  easy  to  get.  The 
conditions  are  undoubtedly  very  complex,  so  that  it  is  perhaps  not  to  be 
expected  that  completely  satisfactory  results  will  be  obtained  until  the 
necessary  conditions  are  better  understood.  Certain  results  have  been 
described  by  A.  E.  Taylor  (155)  and  by  Brailsford  Robertson  (138). 
The  former  obtained  a  synthesis  of  a  protamine  by  the  action  of  trypsin 
on  the  products  of  a  tryptic  digestion  of  the  same  substance,  while  the 
latter  obtained  "  paranuclein  "  by  the  action  of  pepsin  on  the  products  of  a 
prolonged  peptic  digestion  of  caseinogen  ;  but,  considering  the  somewhat 
ill-defined  nature  of  paranuclein  and  the  bodies  related  to  it,  this  latter 
result  cannot  be  regarded  as  of  great  import. 

The  bodies  known  as  "  plasteins  "  are  in  all  probability  to  be  looked 
upon  as  results  of  synthetic  action.  Danilewski  (45)  showed  that  rennet 
preparations  produced  a  precipitate  in  concentrated  solutions  of  Witte's 
peptone,  while  subsequent  workers  found  that  the  same  effect  was  pro- 
duced by  pepsin  and  papain.  These  bodies  are  formed  under  such  con- 
ditions that  a  reverse  reaction  would  be  expected  to  be  most  favoured  ; 
the  amount  found  was  greater  the  higher  the  concentration  of  the  pep- 
tone solution  used.  It  does  not  appear,  however,  that  these  plasteins 
are  necessarily  the  same  as  the  original  protein  from  which  the  peptone 
was  made ;  the  plastein  from  caseinogen  peptone  does  not,  on  acid  hydro- 
lysis, give  quantitatively  the  same  result  in  amino-acids  as  the  casein 
itself.  This  is  not  surprising,  when  we  consider  the  variety  of  possible 
hydrolytic  products. 

A  concentrated  solution  of  products  of  tryptic  digestion  of  caseinogen 
also  shows  a  diminution  of  electrical  conductivity  under  the  action  of 
trypsin,  which  seems  to  indicate  a  synthetic  process. 

Experiments  made  by  Abderhalden  and  Rona  (2,  p,  35)  to  detect 


REVERSIBILITY  OF  ENZYME-ACTION  35 

whether  there  was  any  synthesis  of  polypeptides  from  their  constituent 
amino-acids  under  the  influence  of  tissue  enzymes  led  to  no  result. 

Although  the  direct  evidence  on  the  subject  of  protein-synthesis  is 
at  present  meagre,  the  phenomena  seen  in  trypsin  digests  are  quite  what 
would  be  expected  if  equilibrium  in  a  reversible  reaction  be  the  explana- 
tion of  what  takes  place.  Such  phenomena  are  (i)  retardation  due  to 
accumulation  of  the  products  of  the  reaction,  (2)  recommencement  of  a 
reaction  which  had  apparently  come  to  an  end,  if  the  products  be  re- 
moved by  dialysis,  or  other  means,  or  if  their  concentration  be  reduced 
by  dilution.  In  the  case  of  this  enzyme,  however,  the  products  act 
also  in  another  way  in  diminishing  the  rate  of  change,  namely,  by  re- 
ducing the  alkalinity  of  the  solution,  as  will  be  shown  in  a  later  chapter. 


CHAPTER  VI. 

THE  VELOCITY  OF  REACTION  AND  THE  VARIOUS  CONDITIONS 
AFFECTING  IT. 

THE  function  of  enzymes,  as  catalysts,  being  to  change  the  rate  of  re- 
actions, it  follows  that  the  study  of  their  action,  apart  from  that  of  the 
nature  of  the  products  formed,  consists  essentially  in  the  investigation 
of  the  velocity  of  reactions  and  the  factors  which  have  an  influence  upon 
this. 

In  the  discussion  of  this  problem  a  certain  amount  of  use  must  be 
made  of  mathematical  forms  of  expression.  Since  there  is  a  tendency 
to  decry  the  introduction  of  formulae  into  biological  science,  a  few 
words  are  advisable  upon  the  value  of  such  a  mode  of  treatment. 

Although  it  may  be  perfectly  true  that  by  mathematical  analysis  no 
new  facts  are  discovered,  it  is  none  the  less  true  that  the  expression  of 
experimental  results  in  a  formula  shows  their  relation  to  known  laws  in 
a  way  which  is  otherwise  very  difficult  or  impossible  to  attain.  Further, 
as  Arrhenius  (9,  p.  7),  points  out,  such  a  procedure  enables  one  to  see 
whether  all  the  factors  have  been  taken  into  account ;  instances  of  this 
will  appear  in  the  course  of  the  present  chapter.  Even  an  empirical 
formula  may  assist  in  deciding  whether  irregularities  are  due  merely  to 
experimental  error  or  otherwise. 

The  law  of  mass-action  tells  us  that  a  reaction  proceeds  at  a  rate 
proportional  to  the  concentration  of  the  reacting  molecules.  The 
number  of  times  per  unit  of  time  that  one  molecule  encounters  another, 
with  which  it  can  enter  into  reaction,  is  obviously  related  to  the  number 
present  in  a  given  volume,  that  is,  to  the  concentration.  So  that  when 
the  reaction  can  be  expressed  as  the  change  in  concentration  of  one  kind 
of  molecules,  the  rate  of  change  r  at  a«y  moment  is  proportional  to  the 
amount  of  this  substance  still  left  undecomposed.  Such  a  case  is  the 
hydrolysis  of  saccharose  under  the  action  of  hydrion  in  dilute  solution. 
It  is  true,  of  course,  that  for  each  molecule  of  sugar  inverted  a  molecule 
of  water  is  taken  up,  but  as  the  reaction  is  taking  place  in  excess  of 
water  this  factor  is  not  appreciable.  If  we  call  x  the  amount  of  sugar 
inverted  in  the  time  /,  the  average  rate  of  the  change  during  the  time  t 

x 
is  -,  and  if  C  is  the  concentration  of  saccharose  at  this  time,  the  velocity 

is  proportional  to  it,  /*.£., 

-    =  kC 

k  being  some  constant. 

But,  owing  to  the  continuous  hydrolysis  of  the  sugar,  its  concentra- 
tion (C)  is  not  the  same  at  any  two  consecutive  periods  of  time,  so  that 

36 


THE  VELOCITY  OF  REACTION  37 

the  above  equation  is  only  correct  when  t  is  so  short  that  no  appreciable 
change  has  taken  place  in  the  sugar-content.  This  is  expressed  in  the 
notation  of  the  differential  calculus  thus  :  — 


=kC 
dt 


or,  since  x  is  proportional  to  C, 


the  minus  sign  indicating  that  C  (^concentration  of  saccharose)  is  dim- 
inishing. The  symbols  dx  and  dt  are  to  be  taken  as  wholes,  and  simply 
mean  that  x  and  t  are  to  be  taken  so  small  that  the  velocity  has  not 
changed  during  the  time  /. 

A  reaction  such  as  this,  which  can  be  adequately  treated  as  consist- 
ing of  the  change  of  concentration  of  one  substance,  is  called  a  "  uni- 
molecular  "  reaction  and  is  expressed  by  the  equation  given. 

Now  it  is  clear  that  to  make  any  practical  use  of  the  equation  some 
means  must  be  devised  in  order  to  render  it  applicable  to  data  in  which 
the  time  is  sufficiently  long  to  be  measured  ;  such  a  process  is  known 
as  "integration".  It  is  impossible  in  the  limits  of  this  book  to  describe 
the  method  in  detail,  and  the  reader  is  referred  to  the  Introduction  to 
Mellor's  Chemical  Statics  and  Dynamics  for  further  information. 
Suffice  it  to  say  that  the  process  is  an  artifice  by  which  an  exceedingly 
large  number  of  exceedingly  small  quantities  are  added  together,  so 

dx 
that,  e.g.,  all  the  values  of  -37  during  the  space  of  ten  minutes  are  added 

together.  The  change  of  concentration  in  such  times  can  be  determined 
by  some  one  of  the  methods  previously  described. 

If  it  be  called  to  mind  that  the  kind  of  process  with  which  we  have 
to  deal  is  one  where  the  velocity  at  any  given  moment  depends  on  that 
of  the  moment  preceding,  and  that  such  a  process  when  plotted  out  as  a 
curve  forms  a  logarithmic  curve,  that  is  a  curve  such  that  one  set  of 
co-ordinates  is  a  series  of  numbers  and  the  other  set  the  logarithms  of 
these  numbers,  it  may  help  us  to  understand  why  the  integral  of  our 
differential  equation  has  a  logarithmic  form.  This  integral  may  be 
put  in  various  forms,  but  for  practical  use  the  following  form  is  the  most 
appropriate  one  for  enzyme  work,  in  which  the  initial  and  end  points 
are  apt  to  be  uncertain  :  — 

k  =  -  —  -lognat  p1 

^2  ~    1  2 

where  k  is  the  velocity-constant  of  the  original  equation  and  Q  and  C2 
the  concentrations  of  the  substrate  at  the  times  t^  and  /2  respectively, 
reckoned  from  the  commencement  of  the  reaction.  As  will  be  seen, 
any  two  determinations  during  the  course  of  the  reaction  can  be  used 
for  the  calculation  of  the  value  of  k. 

Another  form,  from  which,  in  fact,  the  above  is  derived  and  which 
is  often  useful,  is 


in  which  t  is  the  time  which  has-  elapsed  since  the  beginning  of  the  re- 
action, a  is  the  initial  concentration  of  the  substrate  and  x  is  the  amount 


38  THE  NATURE  OF  ENZYME-ACTION 

of  products  formed  during  the  time  /,  so  that  a  -  x  is  the  substrate-con- 
centration at  the  end  of  the  time  /. 

The  following  table  will  serve  to  show  the  kind  of  values  obtained 
in  a  unimolecular  reaction,  viz.,  the  inversion  of  cane-sugar  by  acid ;  it 
will  be  seen  that  the  velocity-constant,  as  calculated  by  the  above  equa- 
tion, is  practically  the  same  throughout,  within  the  limits  of  experimental 
error : — 

Time  in  minutes.  Rotation.                       Velocity-constant. 

o  4675°  0-001330 

30  41-00°  1332 

60  3575°  *35* 

90  3°'75°  J379 

120  26-00°  1321 

150  22-00°  1371 

210  15-00°  1465 

33o  2-75°  1463 

510  -  7-00° 

630  —  10-00° 

oo  -I8750 

Now  suppose  that,  instead  of  using  acid,  the  enzyme  invertase  had 
been  employed  and  the  velocity-constant  calculated  by  the  same  formula, 
we  obtain  the  following  results  (V.  Henri  [79,  p.  55]): — 

Time  in  minutes.  Proportion  inverted  (~)              Velocity-constant. 

\&s* 

66  0-084  0-00058 

168  0-220  64 

334  °'426  72 

488  0-581  77 

696  0-746  85 

1,356  0-952  97 

The  constant  shows  a  steady  rise. 

Take  now  a  corresponding  series  of  values  from  the  experiments  of 
Frankland  Armstrong  (5,  ii.,  p.  506)  on  the  hydrolysis  of  milk-sugar  by 
lactase : — 

Time  in  hours.  Velocity-constant. 

1  0-0640 

2  0-0543 

3  0-0460 
5  0-0310 

24  0*0129 

In  this  case,  unlike  that  of  invertase,  there  is  a  steady  fall  in  the  values 
of  the  velocity-constant. 

Another  case  is  that  of  trypsin,  the  following  table  being  taken  from 
an  experiment  of  my  own  :— 

ist    10  minutes  £  =  0-0079 

2nd         „  0-0046 


3rd 
4th 
5th 
7th 
gth 


0*0032 

0*0022 

0-0016 
0*0009 
0-0007 


Here,  again,  there  is  a  marked  diminution  in  the  values  of  £as  calculated 
by  the  simple  unimolecular  formula. 

What  is  the  explanation  of  these  disagreements  with  the  said  law  ? 

In  the  first  place  it  must  be  noted  that  cases  in  which  the  velocity 
is  greater  at  any  given  time  than  that  calculated  by  the  unimolecular 
formula  are  unusual ;  we  will  therefore  consider  first  the  opposite  case, 
which  is  that  found  to  apply  to  most  enzymes. 


THE  VELOCITY  OF  REACTION 


39 


The  contrast  between  the  three  cases  dealt  with  will  be  made  clearer 
if  they  are  put  in  the  form  of  curves,  as  is  done  in  Fig.  2. 

In  this  figure  curve  A  is  that  of  the  action  of  invertase,  taken  from 
one  of  Victor  Henri's  experiments;  the  ordinates  are  given  by  the 
numbers  on  the  left  of  the  figure  and  represent  proportions  per  cent, 
inverted ;  curve  B  is  the  true  logarithmic  one  of  acid  inversion,  and  curve 
C  is  that  of  the  changes  in  electrical  conductivity  in  the  action  of  trypsin 
on  caseinogen,  taken  from  one  of  my  own  experiments,  the  ordinates 
being  given  in  gemmhos  (  =  reciprocal  megohms)  on  the  right  of  the 
figure.  The  numbers  on  the  axis  of  abscissae  denote  hours  and  apply 
to  all  the  curves. 

It  is  found  by  experiment  that  in  many  cases  the  enzyme  itself  dis- 
appears in  the  course  of  its  action.  Since  the  amount  of  the  catalysis 
is  always  in  direct  proportion  to  the  concentration  of  the  catalyst,  al- 
though, as  we  shall  see,  not  always  in  linear  proportion,  a  gradual  de- 


6 


8        9       10 


FIG.  2. 


struction  of  the  enzyme  would  lead  to  a  slowing  of  the  reaction  greater 
than  that  due  to  the  diminished  concentration  of  the  substrate.  We 
have  already  seen  that  in  fairly  pure  solution  most  enzymes  are  unstable, 
especially  at  a  temperature  of  38°,  but  that,  on  the  other  hand,  in  the 
presence  of  their  substrates  or  products  they  are  much  more  stable. 
Trypsin  is  one  of  the  most  unstable  of  enzymes  when  these  protecting 
influences  are  not  present;  Vernon  (162,  p.  378)  found  that  70  per  cent, 
was  destroyed  in  0*4  per  cent,  sodium  carbonate  solution  in  an  hour. 
However,  in  an  actual  digestion  mixture  I  found,  in  a  series  of  experi- 
ments made  in  the  manner  given  by  V.  Henri  (80,  pp.  302-303),  that  no 
diminution  of  the  activity  of  the  enzyme  could  be  detected  up  to  six 
hours  in  the  conditions  of  the  experiment  from  which  the  data  of  the 
table  above  were  obtained,  and  very  little  up  to  seven  hours.  The 
method  was  as  follows :  in  order  to  have  all  the  other  conditions,  with 


40  THE  NATURE  OF  ENZYME-ACTION 

exception  of  the  enzyme,  constant,  at  various  stages  in  the  course  of  a 
series  of  determinations  of  the  electrical  conductivity  of  a  reacting  mix- 
ture of  caseinogen  and  a  known  initial  concentration  of  trypsin,  samples 
were  removed  and  immediately  immersed  in  small  flasks  in  boiling  water, 
in  order  to  destroy  the  enzyme.  To  each  of  these  trypsin  was  then  added 
in  the  amount  requisite  to  make  the  concentration  the  same  as  it  was  at  the 
beginning;  the  rate  of  change  was  now  compared  with  that  of  the 
original  solution  at  the  same  stage.  If  the  samples  with  fresh  enzyme 
showed  a  greater  rate  of  change  than  the  main  digest  at  the  same  stage, 
it  is  obvious  that  there  was  less  enzyme  present  in  the  latter  than  had 
been  originally  put  in,  since  all  other  conditions  were  identical.  The 
results  showed  that  the  destruction  of  enzyme  was  not  the  chief  cause 
of  the  falling  off  of  activity,  since  there  was  no  appreciable  destruction 
up  to  eight  hours.  Similar  results  were  obtained  by  Tammann  in  the 
case  of  emulsin. 

There  is  then  some  other  cause  for  the  deviation  from  the  logarith- 
mic law.  This  is  found,  by  experiment,  to  consist  in  the  effect  of  the 
products  of  the  reaction.  When  these  are  added  to  a  reacting  mixture, 
a  change  in  the  velocity  of  the  reaction  occurs  such  as  shows  itself  when 
the  same  products  make  their  appearance  during  the  normal  course  of 
the  reaction. 

There  are  several  ways  in  which  this  effect  of  the  products  of  the 
reaction  shows  itself.  In  the  first  place,  there  is  abundant  evidence  that 
a  combination  of  some  kind  is  formed  between  the  enzyme  and  the 
substrate  preparatory  to  the  action  of  the  former.  There  is  also  a  similar 
combination  between  the  enzyme  and  products,  as  would  naturally  be 
expected,  if  both  hydrolytic  and  synthetic  processes  are  catalysed ;  if 
combination  between  enzyme  and  substrate  is  requisite  for  the  former, 
then  presumably  combination  between  enzyme  and  products  is  requi- 
site for  the  latter.  But  let  us  see  what  evidence  there  is  of  such  com- 
binations of  either  kind. 

As  long  ago  as  1806  Clement  and  Desormes  (43)  put  forward  one 
of  the  first  explanations  of  a  catalytic  process  in  their  theory  of  the 
action  of  nitrous  acid  in  the  oxidation  of  sulphur  dioxide  to  sulphuric 
acid.  This  explanation  consisted  in  the  formation  of  a  temporary 
combination  between  the  substrate  and  catalyst  in  the  shape  of  what  is 
now  called  nitrosulphonic  acid,  which  afterwards  decomposed  with 
formation  of  sulphuric  acid  and  regeneration  of  the  catalyst.  The  actual 
proof  of  such  intermediate  combinations  has  only  been  given  in  more 
recent  times  by  Erode  (36),  as  already  mentioned.  A  similar  course  of 
events  is  also  now  regarded  as  the  most  acceptable  theory  of  the  action 
of  enzymes. 

The  enzyme  appears  as  a  rule  to  enter  into  a  state  of  association 
with  some  particular  molecular  grouping  in  the  substrate,  so  that,  if  this 
is  an  uncommon  grouping,  the  enzyme  will  be  very  "specific  "  ;  thus  in- 
vertase  enters  into  relation  only  with  fructose,  maltase  only  on  bodies 
having  the  structure  of  the  a-glucosides  and  so  on.  Such  an  intimate 
correlation  is  particularly  well  seen  in  the  case  of  the  various  yeast- 
enzymes  acting  on  disaccharides,  which  were  investigated  by  Emil 
Fischer,  and  which  led  him  to  formulate  his  famous  simile  of  the  "  lock- 
and-key  "  relationship  (63,  p.  2992).  This  implies  a  very  close  similarity 
in  configuration  between  enzyme  and  substrate,  and  since,  as  we  have 


THE  VELOCITY  OF  REACTION  41 

seen,  it  applies  also  to  optical  opposites,  it  makes  it  probable  that  the 
enzymes  in  question  are  themselves  optically  active.  There  is,  moreover, 
evidence  that  other  enzymes  are  optically  active.  The  work  of  Dakin 
(44)  on  hydrolysis  of  optically  active  esters  by  the  lipase  of  liver  affords 
evidence  on  this  point  as  well  as  on  the  general  question  of  combination. 
It  was  found  that,  when  the  optically  inactive  mixture  of  the  two  esters 
of  mandelic  acid  was  acted  on  by  this  enzyme,  the  dextro-component 
was  hydrolysed  more  rapidly  than  the  laevo-component.  In  this  way  it 
happened  that  the  percentage  of  dextro-mandelic  acid  in  the  products 
became  greater  at  first  than  that  of  the  Isevo-acid,  so  that  the  mixture 
was  optically  active ;  as  the  reaction  proceeded  the  relative  amounts 
approximated  more  and  more,  until  finally  both  were  present  in  equal 
quantity  and  the  mixture  became  again  optically  inactive.  These  facts 
can  only  be  satisfactorily  explained  on  the  hypothesis  that  the  enzyme 
itself  is  optically  active  and  forms  addition  compounds  with  the  esters. 
As  Dakin  puts  the  matter  :  "  The  dextro-  and  laevo-components  of  the 
inactive  ester  first  combine  with  the  enzyme,  but  the  latter  is  assumed 
to  be  an  optically  active  asymmetric  substance,  so  that  the  rates  of  com- 
bination of  the  enzyme  with  the  d-  and  1-esters  are  different.  The 
second  stage  in  the  reaction  consists  in  the  hydrolysis  of  the  complex 
molecules  of  (enzyme  +  ester).  Since  the  complex  molecule  (enzyme 
+  d-ester)  would  not  be  the  optical  opposite  of  (enzyme  +  1-ester),  the 
rate  of  change  in  the  two  cases  would  again  be  different.  Judging  by 
analogy  with  other  reactions  one  might  anticipate  that  the  complex 
molecule  which  is  formed  with  the  greater  velocity  would  be  more 
rapidly  decomposed.  In  the  present  case  it  would  appear  that  the 
dextro-component  of  the  inactive  mandelic  ester  combines  more  readily 
with  the  enzyme  than  the  laevo-component  does,  and  that  the  complex 
molecules  (d-ester  +  enzyme)  are  hydrolysed  more  rapidly  than  (1- 
ester  +  enzyme),  so  that  if  the  hydrolysis  be  incomplete  dextro-acid  is 
found  in  solution  and  the  residual  ester  is  laevo-rotatory."  I  would,  in 
passing,  call  attention  to  the  fact  that  in  these  experiments,  unlike  the 
case  of  emulsin  acting  on  the  methyl  glucosides,  the  lipase  does  not 
show  itself  to  be  capable  of  acting  on  one  of  the  two  optical  isomers 
only;  it  hydrolyses  both  but  at  unequal  rates.  This  circumstance, 
on  the  face  of  it,  looks  more  like  a  kind  of  combination  approxi- 
mating to  a  physical  type  rather  than  to  chemical  union  in  the  strict 
sense.1 

That  there  is  a  marked  "affinity  "  for  certain  optically  active  groups 
is  shown  by  the  work  of  Fischer  and  his  coadjutors  (65),  Abderhalden 
and  others,  on  the  relation  of  trypsin  to  di-  and  polypeptides.  With- 
out entering  into  details,  it  must  suffice  to  say  here  that  it  is  impossible 
at  present  to  give  any  general  rule  as  to  which  of  these  compounds  is 
hydrolysed  by  trypsin  ;  only  compounds  of  naturally  occurring  amino- 
acids  are  attacked,  and  amongst  these  there  is  a  preference  shown  for 
those  containing  tyrosine  or  leucine  and,  in  a  somewhat  less  degree,  for 
those  containing  alanine.  At  the  same  time,  many  curious  preferences 
are  to  be  observed,  for  example,  alanyl-glycine  is  hydrolysed,  but  glycyl- 
alanine  is  not,  1-leucyl-l-leucine  is  attacked,  but  neither  1-leucyl-d-leucine 
nor  d-leucyl-1-leucine  is,  and  so  on.  For  further  details  as  to  this 

ASee  Note  D. 


42  THE  NATURE  OF  ENZYME-ACTION 

problem,  the  monograph  on  the  amino-acids  by  Dr.  Aders  Plimmer,  in 
this  series,  must  be  consulted. 

Further  evidence  upon  the  combination  of  enzyme  and  substrate 
was  afforded  by  the  observation  of  O'Sullivan  and  Tompson  (129) 
in  1890.  They  found  that  invertase  will  withstand  uninjured  a 
temperature  25°  higher  in  the  presence  of  cane-sugar  than  in  its 
absence.  As  they  point  out,  it  is  difficult  to  see  how  this  could  hap- 
pen unless  the  enzyme  entered  into  some  kind  of  union  with  the 
sugar. 

Another  phenomenon,  which  is  impossible  to  explain  except  on  the 
hypothesis  of  a  combination  of  this  kind,  is  the  law  of  the  reaction- 
velocity  in  the  initial  stages  of  certain  enzyme-actions  when  low  con- 
centrations of  the  enzyme  are  used.  Duclaux  (50,  52)  found  that, 
under  these  conditions,  the  rate  of  change,  in  the  case  of  invertase,  did 
not  follow  the  law  of  mass-action,  but  that  the  amount  of  cane-sugar 
inverted  was  directly  proportional  to  the  time  of  action,  or,  in  other 
words,  that  the  same  quantity  was  hydrolysed  in  the  second  ten 
minutes  as  that  hydrolysed  in  the  first  ten  minutes ;  the  curve  instead 
of  being  logarithmic,  became  a  straight  line.  In  1902  Adrian  Brown 
(37)  showed  that,  when  cane-sugar  solutions  of  varying  concentrations 
were  hydrolysed  under  the  influence  of  invertase,  in  the  early  stages  of  the 
reaction  the  amount  inverted  in  equal  times  was  nearly  the  same  in  all. 
According  to  the  law  of  mass-action  these  amounts  should  have  been 
proportional  to  the  concentrations  of  the  substrate.  In  order  to  explain 
this  result,  Adrian  Brown  assumed  that  not  only  is  there  formed  a 
compound  of  enzyme  and  sugar,  but  that  this  exists  for  an  appreciable 
time ;  consequently  a  definite  quantity  of  the  enzyme  can  only  effect  a 
limited  number  of  complete  molecular  changes  in  a  given  time ;  what- 
ever the  available  mass  of  the  substrate  may  be,  if  it  is  greater  than  the 
amount  of  enzyme  which  is  present  and  with  which  it  can  enter  into 
combination,  no  increase  in  the  amount  changed  is  possible.  If  the 
ratio  of  the  enzyme  to  the  cane-sugar  be  greater  than  a  certain  value 
the  amount  of  the  latter  hydrolysed  is  directly  proportional  to  the  con- 
centration, as  the  logarithmic  law  requires. 

This  question  is  further  discussed  in  a  paper  by  Horace  Brown  and 
Glendinning  (39)  on  the  relations  of  starch  and  amylase.  They  point 
out  that,  when  the  concentration  of  the  enzyme  is  very  small  relatively 
to  that  of  the  starch,  in  the  early  stages  of  the  reaction,  as  long  as  this 
excess  of  substrate  remains  unhydrolysed,  the  amount  of  starch  per  unit 
volume  will  be  very  large  compared  with  the  amount  of  the  combina- 
tion of  starch  and  enzyme.  So  long,  therefore,  as  the  concentration  of 
the  unchanged  substrate  remains  very  large  in  relation  to  that  of  the 
combination,  the  latter  will  remain  nearly  constant  in  amount  and 
equal  amounts  of  starch  will  be  hydrolysed  in  equal  times,  the  curve 
being  a  straight  line.  Subsequently,  when  the  concentration  of  the 
starch  has  been  much  reduced,  the  amount  of  the  combination  and 
consequently  the  hydrolysis  of  the  sugar  will  follow  more  closely  the 
law  of  mass-action.  '  It  is  pointed  out  also  that  this  explanation  is  in 
agreement  with  experimental  facts. 

Similar  results  were  obtained  by  Frankland  Armstrong  (5,  ii.,  p. 
508)  in  his  work  on  lactase,  maltase  and  emulsin.  The  following 
numbers  give  the  amounts  of  milk-sugar  hydrolysed  in  forty-six  hours 


THE  VELOCITY  OF  REACTION 


43 


by  a  very  small  amount  of  lactase  acting  on  different  strengths  of  the 
solution  of  sugar  : — 


Solutions 
containing 

Proportion  Hydrolysed. 

Actual  Weight. 

10  per  cent. 

20 

30 

22'2 

10-9 

77 

2'22 

2-18 

2'2I 

Experiments  in  which  the  proportion  of  enzyme  present  was  large 
relatively  to  the  concentration  of  the  sugar  gave  a  different  result. 


Milk-sugar  per 

100  C.C. 

Amount  Changed  in 
Three  Hours. 

Velocity-constant. 

i-o  gramme 
o'5 

0-2 

0-185 
0-098 
0-0416 

0-0296 
0-0298 
0-0337 

The  amount  hydrolysed  was  in  exact  ratio  to  the  concentration  of  the 
sugar,  while  the  velocity-constant  was  nearly  the  same  in  all. 

In  some  cases,  as  mentioned  previously,  such  as  those  of  emulsin  on 
amygdalin  and  trypsin  on  caseinogen,  it  has  been  noticed  that,  when 
the  reaction  appears  to  be  at  an  end,  the  addition  of  more  substrate 
causes  a  further  progress  of  the  reaction,  which  shows  that  the  enzyme 
was  in  some  way  bound  up  in  the  constituents  of  the  equilibrium. 
That  the  cessation  of  change  was  not  due  to  exhaustion  of  the  substrate 
is  shown  by  the  fact  that  addition  of  more  enzyme  also  produced  a 
further  hydrolysis. 

When  we  come  to  examine  the  evidence  for  combination  between 
enzyme  and  products  it  is  found  that  it  is  of  a  similar  nature  to  that 
just  dealt  with,  but  perhaps  less  direct. 

O'Sullivan  and  Tompson  (129),  in  the  work  above  mentioned, 
showed  that  invertase  was  protected  from  the  action  of  heat  by  pro- 
ducts of  the  inversion  of  cane-sugar,  as  well  as  by  the  sugar  itself. 
Trypsin  is  much  more  stable  in  the  presence  of  either  substrate  or  pro- 
ducts than  alone.  This  was  shown  for  peptone  by  Starling  and  myself 
(i  8)  and  for  amino-acids  by  Vernon  (163,  p.  354). 

There  is  no  doubt  that,  in  the  case  of  the  sucroclastic  enzymes,  as 
investigated  by  Frankland  Armstrong  (5,  iii.,  p.  520),  there  is  a  special 
retarding  influence  exerted  by  the  respective  products  of  the  enzymes, 
lactase,  emulsin,  maltase  and  invertase,  on  the  rates  of  hydrolysis  by 
these  enzymes,  an  effect  which  is  not  shown  by  other  sugars.  For  ex- 
ample, fructose  retards  invertase,  but  has  no  effect  on  any  one  of  the 
other  enzymes,  galactose  has  very  little  effect  on  maltase,  but  consider- 
able retarding  action  on  lactase. 

Now  these  results  can  partly  be  explained  by  the  reversibility  of  the 
reactions.  If  we  examine  the  data  given  by  the  author  we  notice  that, 
while  both  of  the  products  have  an  action  of  the  kind  in  question,  the 
effect  of  one  is  usually  more  marked  than  that  of  the  other.  This  was 
believed  to  be  due  to  the  fact  that  the  enzyme  in  each  case  has  the  pro- 


44 


THE  NATURE  OF  ENZYME-ACTION 


perty  of  combining  in  a  special  manner  with  a  particular  sugar,  and  by 
this  means  is  withdrawn  from  the  sphere  of  action.  An  interesting  fact 
in  this  connection  is  that  the  action  of  emulsin  on  milk-sugar  is  retarded 
by  a-methylglucoside  which  is  not  attacked  by  the  enzyme,  showing 
that  an  enzyme  is  able  to  form  some  kind  of  union  with  a  sugar 
although  it  may  not  be  close  enough  to  hydrolyse  it.  From  the  con- 
sideration of  relations  of  this  kind,  it  appears  that  these  sugar-splitting 
enzymes  enter  into  relation  with  their  substrates  along  nearly  the  whole 
of  the  molecule,  but  that  a  small  degree  of  misfit,  so  to  speak,  prevents 
actual  hydrolysis.  To  take  the  cases  of  the  two  methylglucosides  and 
their  relations  to  maltase  and  emulsin  respectively,  the  state  of  affairs 
may  be  represented  by  the  diagrams  below  (Fig.  3),  in  which  it  will  be 
seen  that  emulsin,  for  example,  is  only  "out  of  harmony"  with  the  a- 
glucoside  at  the  extreme  top  of  the  figure,  yet,  as  we  know,  this  is 
sufficient  to  prevent  its  action.  It  must  be  understood  that  these  figures, 


5  \ 

H         H( 
i'               i 

)N\ 

:  ^ 

Hv  ,0^«3    ( 

)H 

J       C 

//' 

R 

/              H 

\ 

'      \ 

\H         \ 

i 

» 
i 
i 

x  C 
H 

—           v 

.,—          —  \j 
H 

IK 

;OH 

H( 

:OH 

HCOH 
H 

HCOH 
H 

FIG.  3. 

being  only  in  one  plane,  cannot  represent  the  real  shape  of  the  molecules, 
so  that  they  must  be  taken  merely  as  a  kind  of  shorthand  to  express 
the  experimental  facts. 

Results  of  a  similar  nature  were  obtained  by  Abderhalden  and  Gigon 
(i)  in  the  case  of  the  action  of  yeast  press-juice  on  glycyl-l-tyrosine. 
The  addition  of  the  amino-acids  d-alanine,  d-valine,  1-leucine,  1-tyrosine, 
tryptophane  and  d-glutamic  acid,  which  are  constituents  of  polypeptides 
hydrolysed  by  the  enzyme,  were  found  to  retard  the  reaction  ;  but  1- 
alanine  and  d-leucine  have  no  effect.  In  other  words,  amino-acids, 
which  are  found  in  bodies  upon  which  the  enzyme  acts,  are  able  to  enter 
into  a  combination  of  such  a  kind  with  the  enzyme  that  this  is  with- 
drawn from  the  reacting  system. 

Tammann  (152)  showed  that  the  action  of  emulsin  on  amygdalin 
was  retarded  by  any  of  the  products  of  reaction,  and  I  have  myself  re- 
cently found  that  the  action  of  the  same  enzyme  on  arbutin  (hydroqui- 


THE  VELOCITY  OF  REACTION  45 

none-glucoside)  is  retarded  by  both  glucose  and  by  hydroquinone  and, 
indeed,  to  very  nearly  the  same  degree  by  both.  A  O'2N  solution  of 
arbutin  was  hydrolysed  by  emulsin  at  37 '5°  to  the  extent  of  26*6  per 
cent,  in  twenty-five  hours ;  a  similar  solution,  to  which  hydroquinone 
had  been  added  to  an  amount  such  as  to  make  the  solution  0*1  N,  showed 
a  change  of  only  1 5  per  cent. ;  another  similar  one,  with  glucose  in  place 
of  hydroquinone,  showed  13*5  per  cent,  hydrolysis,  while  a  fourth  which 
was  0*05  N  in  both  glucose  and  hydroquinone  was  hydrolysed  to  13*25 
per  cent.  Considering  the  great  number  of  different  glucosides  which 
are  attacked  by  emulsin  it  is  very  difficult  to  believe  that  the  enzyme  is 
able  to  enter  into  relation  with  the  non-sugar  part  of  all  these  bodies, 
which  have  such  varied  chemical  constitution.  If  the  phenomena  can  be 
explained  in  another  way  it  is  obviously  to  be  preferred. 

So  far  as  we  know,  all  the  reactions  catalysed  by  enzymes  are  revers- 
ible, differing  only  as  to  the  position  of  equilibrium  ;  in  some  instances, 
as  that  of  invertase,  the  equilibrium-point  is  so  near  that  of  complete 
hydrolysis  that  it  needs  careful  investigation  to  detect  that  the  latter  is 
not  quite  complete.  To  return  for  a  moment  to  the  typical  case  of 
acid,  alcohol  and  ester,  represented  by  the  equation — ester  +  water  ^ 
acid  +  alcohol,  let  us  suppose  that  we  start  with  ester  and  water  alone, 
with  a  lipase  as  a  catalyst.  At  the  commencement,  the  reaction  of 
hydrolysis  will  be  rapid,  while  the  reverse  reaction  of  ester  formation 
will  be  zero ;  as  soon,  however,  as  any  perceptible  amount  of  acid  and 
alcohol  are  formed,  the  synthetic  reaction  will  begin,  at  first  very  slowly 
but  gradually  gaining  in  rate  as  the  concentration  of  the  hydrolytic 
products  increases,  while  the  hydrolytic  reaction  will  become  slower  as 
the  concentration  of  the  substrate  diminishes,  until  finally  the  rates  of 
the  two  opposite  reactions  are  equal  and  equilibrium  is  established. 
Now  if  we  direct  our  attention  to  the  hydrolytic  process  alone,  we  see 
that  it  is  more  and  more  counteracted  by  the  opposite  process  as  the 
reaction  proceeds,  so  that  the  actual  values  obtained  in  an  experiment 
are  really  the  differences  between  two  opposite  reactions.  As  Benjamin 
Moore  (120,  p.  79)  rightly  points  out,  this  reverse  reaction  will  show  itself 
as  a  slowing  of  the  hydrolytic  process  to  a  greater  and  greater  degree  as 
equilibrium  is  approached,  even  in  those  cases  where  the  hydrolysis  is 
practically  complete. 

The  actual  velocity  of  reaction  at  any  moment  is  then  to  be  regarded 
as  the  difference  between  two  opposite  processes.  We  see  also  why 
either  of  these  reactions  can  be  accelerated  by  the  addition  of  substrate 
or  products  respectively. 

It  will  be  remembered  that  Croft  Hill  showed  that  the  equilibrium 
in  the  case  of  maltase  was  a  genuine  one,  inasmuch  as  the  same  point 
was  reached  starting  from  either  end,  glucose  or  the  disaccharide.  There 
are  also  two  important  researches  in  which  the  two  opposite  velocity- 
constants,  whose  ratio  gives  the  equilibrium,  have  been  determined ; 
namely  that  of  Visser  on  invertase  and  emulsin,  and  that  of  Dietz  on 
lipase,  both  of  which  have  been  already  mentioned  in  other  connections. 
It  is  necessary  now  to  consider  these  results  in  somewhat  more  detail. 
We  will  take  first  the  hydrolysis  of  salicin  by  emulsin.  As  it  was 
shown  by  Tammann  that  the  action  of  this  enzyme  was  retarded  by 
the  products  of  its  activity,  it  was  advisable  to  see  whether  it  could  be 
shown  to  produce  salicin  from  the  hydrolytic  products,  viz.,  glucose  and 


46  THE  NATURE  OF  ENZYME-ACTION 

saligenin.  This  was  found  to  take  place  (164,  p.  276).  Evidence  was 
also  obtained  that  cane-sugar  was  similarly  formed  by  invertase  from 
glucose  and  fructose  (164,  p.  275). 

When  the  velocity-constants  for  the  hydrolytic  process  in  the  two 
cases  are  calculated  from  a  formula  which  takes  account  of  the  two 
opposite  reactions,  it  is  found  that  these  constants,  just  as  when  the 
reverse  reaction  is  disregarded,  still  show  a  considerable  amount  of  the 
same  regular  increase  or  decrease  of  the  values  of  the  velocity-constants 
during  the  progress  of  the  reaction.  It  is  evident,  therefore,  that  some 
other  cause  is  also  present.  Visser  (164,  p.  283)  introduces  the  con- 
ception of  "  intensity "  of  action  of  the  enzyme,  and,  by  introducing 
appropriate  factors  in  the  formula,  it  was  found  that  satisfactory  regu- 
larity of  the  velocity-constants  was  obtained,  even  when  the  reverse 
reaction  was  neglected  altogether  (164,  p.  296).  We  shall  see  presently 
what  meaning  is  to  be  attached  to  this  "  intensity-factor".  It  is  perhaps 
not  surprising  that  the  reverse  reaction  has  comparatively  little  influence 
on  the  rate  of  hydrolysis  in  the  two  instances  dealt  with,  for,  since  the 
equilibrium-position  is  so  near  to  that  of  complete  hydrolysis,  the  velocity 
of  the  synthetic  reaction  must  be  very  small;  some  approximate  nu- 
merical values  have  been  given  in  Chapter  V. 

The  important  researches  of  Dietz  (48)  were  concerned  with  the 
action  of  pancreatic  lipase.  In  order  to  avoid  complications  due  to 
reactions  taking  place  in  steps,  only  univalent  alcohols  and  monobasic 
acids,  with  the  corresponding  esters,  were  used.  All  the  chief  experi- 
ments were  done  with  isoamyl  alcohol  and  normal  butyric  acid.  Since 
the  alcohol  and  water  were  always  in  considerable  excess,  usually  some- 
where near  five  molecules  of  water  to  eight  molecules  of  alcohol,  both  the 
hydrolysis  and  synthesis  could  be  treated  as  unimolecular  reactions  and 
the  calculations  thus  simplified.  In  order  also  to  obtain  the  exact 
equilibrium  the  experiments  were  nearly  always  carried  out  from  both 
sides  simultaneously,  on  the  one  side  a  solution  of  ester  in  water  +  amyl 
alcohol  was  taken  and  on  the  other  side  an  equally  concentrated  solution 
of  butyric  acid  in  amyl  alcohol  +  water. 

The  first  thing  to  notice  is  that  the  equilibrium  position,  as  shown  by 
the  final  concentration  of  acid,  is  the  same  whether  approached  from 
the  side  of  ester  or  from  that  of  acid  (48,  pp.  302-306).  It  was  found, 
moreover,  that,  when  enzyme  preparations  of  different  activity,  as  shown 
by  the  velocity-constants,  were  taken,  the  equilibrium  was  the  same  in 
all.  Similarly,  different  amounts  of  the  same  enzyme  preparation  were 
without  effect  on  this  point.  It  may  be  remarked  here  that  similar 
results  were  obtained  by  Visser  with  invertase  and  emulsin. 

When  experiments  were  made  with  different  initial  concentrations 
of  the  substrates,  results  were  obtained  which  differed  from  what  the 
law  of  mass-action  demands.  The  probable  reason  of  this  will  be  given 
in  the  next  chapter  of  the  present  work. 

As  regards  the  velocity-constants  themselves,  it  was  found  that 
when  the  water-concentration  of  the  butyric  acid  solution  was  low,  the 
ester  formation  followed  the  logarithmic  law  of  a  simple  reaction  of 
the  first  order  as  would  be  expected ;  the  rate  of  the  reverse  reaction, 
the  ester  hydrolysis,  was  therefore  negligible. 

On  proceeding  to  higher  concentrations  of  water,  it  is  seen  that,  if 
butyric  acid  is  the  starting-point,  a  considerable  part  of  it  is  not  esterified, 


THE  VELOCITY  OF  REACTION 


47 


while,  if  the  ester  is  the  starting-point,  it  is  partially  hydrolysed.  In 
this  way  the  two  velocity-constants  can  be  measured.  When  this  is  done 
by  the  regular  unimolecular  formula,  constant  values  in  each  case  are 
obtained,  within  the  limits  of  experimental  error.  The  table  below  is 
a  copy  of  one  of  those  given  by  Dietz  (48,  p.  305) ;  t  =  time  in  hours, 
T  =  millimols  of  acid  per  litre  required  to  neutralise,  kl  =  velocity- 
constant  of  the  synthetic  process  and  £2  =  that  of  the  hydrolytic  process. 
The  reactions  took  place  in  amyl  alcohol  containing  8  per  cent,  of 
water.  ^ 


Ester  Formation: 

t. 

T. 

$ 

0*00 

197-70 

_ 

1-58 

187-40 

0-015 

4-00 

177-60 

0-OI2 

7-00 

160-80 

0-OI3 

10-40 

147-00 

0*013 

I5'05 

126-50 

O'OI4 

24-48 

98-51 

0-OI4 

31-62 

88-06 

0-OI4 

96-30 

48-5I 



CO 

45-90 

0-014  in  the  mean. 

Ester  Hydrolysis. 

t. 

T. 

4 

O'OO 

O'OO 

2'95 

6-86 

0-0055 

7-20 

i3'44 

0-0049 

16-40 

24-25 

0-0046 

23-65 

30-23 

0-0045 

45-07 

40-30 

0-0047 

88-83 

44-40 

CO 

45-90 

0-0048  in  the  mean. 

Fig.  4  will  serve  to  give  a  general  idea  of  the  course  of  the  change  in 
these  experiments.  The  ordinates  represent  the  concentration  of  butyric 
acid  and  the  abscissae  time  in  hours,  so  that  the  upper  curve  A  is  that 
of  ester  hydrolysis  and  the  lower  curve  B  that  of  ester  formation. 
Curve  C  is  that  which  would  be  given  if  the  reaction  went  to  completion 
in  one  direction. 

It  will  be  remembered  that  the  equilibrium  condition  is  definable  in 
two  ways,  either  as  the  ratio  of  the  concentrations  of  the  bodies  taking  part 
in  it,  in  the  present  case  ester  and  acid,  or  as  the  ratio  of  the  two  opposite 
velocity -constants.  From  the  data  given  in  the  tables  above  it  is  possible 
to  obtain  values  in  both  ways.  Calculated  from  the  relative  concentra- 
tions, it  works  out  to  be  3*3  and  from  the  velocity-constants  3*4,  a  very 
satisfactory  agreement.  It  is  to  be  noted,  however,  that  such  good 
agreement  was  not  always  found. 

A  rather  considerable  time  has  been  spent  over  the  results  pf  Dietz, 


48 


THE  NATURE  OF  ENZYME-ACTION 


since  they  form  an  analysis  of  a  fairly  simple  case  and  will  serve  as  a 
foundation  on  which  further  complications  may  be  added. 

One  of  these  complications  is  the  factor  called  by  Visser  the  "  inten- 
sity "  of  the  enzyme,  which  plays  an  important  part  in  many  reactions, 
but  appears  in  the  case  of  lipase  not  to  affect  the  form  of  the  expression 
for  the  reaction-velocity.  The  simplest  way  in  which  this  factor  can  be 
altered  is  plainly  by  variations  in  the  concentration  of  the  enzyme  added. 
As  we  have  seen,  the  rate  of  action  is  proportional  to  this  concentration, 
so  that  if,  during  the  progress  of  a  reaction,  anything  happened  which 
lessened  the  effective  concentration  of  the  enzyme,  the  reaction  would  be 
slowed.  The  combination  of  enzyme  with  its  products  is,  in  fact,  one 
such  circumstance,  as  it  acts  by  removing  enzyme  from  the  sphere  of 
action.  But  the  activity  of  the  enzyme  may  be  affected  in  other  ways 
without  being  actually  removed.  Trypsin,  for  example,  is  extraordin- 
arily sensitive  to  the  presence  of  alkali  (hydroxidion) ;  it  is  practically 


200 

15       30       45       60      75       90     105     120     135     150 

FIG.  4. 

inert  in  acid  or  neutral  solution,  but  is  greatly  assisted  by  the  presence 
of  alkali,  and  up  to  certain  limits  in  direct  ratio  to  the  concentration  of 
this  latter  as  the  following  numbers  show  :  A  series  of  solutions  con- 
taining 2' 5  per  cent,  of  caseinogen  and  the  concentrations  of  ammonia 
shown  in  the  first  column  were  acted  on  by  trypsin  for  one  hour ;  the 
rises  of  conductivity  given  in  the  other  column  were  found. 

Concentration  of  Ammonia.  Change. 

0-05  normal  *>77u  gemmhos 

0-03      „  1,365 

O'OI         ,,  680  ,, 

In  the  hydrolysis  of  proteins  by  trypsin,  as  is  well  known,  a  number 
of  amino-acids  are  set  free,  and  some  of  these,  aspartic  and  glutamic 
acids,  are  fairly  strong  acids,  which  will  considerably  reduce  the  concen- 
tration of  hydroxidions  in  the  solution.  In  fact,  as  I  have  recently  had 
occasion  to  observe,  if  the  initial  alkali-content  was  not  very  large,  say, 


THE  VELOCITY  OF  REACTION  49 

2  c.c.  of  '880  ammonia  to  the  litre  of  10  per  cent,  caseinogen,  the  digest 
becomes  actually  acid  to  litmus.  This  factor  must  then  play  an  import- 
ant part  in  the  slowing  off  of  the  rate  of  change  in  such  a  case. 

In  the  case  of  invertase  there  is  some  influence  at  work  which  has 
the  effect  of  accelerating  the  rate  of  the  reaction  as  it  progresses,  since 
the  velocity-constants  calculated  by  the  logarithmic  formula  steadily 
increase,  as  shown  by  Victor  Henri  (78).  The  explanation  seems  to 
lie  in  some  observations  by  Kullgren  (106),  who  showed  that  a  similar 
rise  in  the  velocity-constants  took  place  in  the  inversion  of  cane-sugar 
by  water  at  1 00° ;  in  this  case  the  rise  was  due  to  the  production  of  an  acid 
as  a  bye-product,  which  would  increase  the  rate  of  hydrolysis.  It  has 
not  been  shown  as  yet  whether  in  the  case  of  invertase  there  is  any  such 
production  of  acid,  although  it  is  not  improbable.  Since  the  action  of 
the  enzyme  is  favoured  by  small  amounts  of  acid,  such  a  production 
would  explain  the  increase  of  "  intensity "  of  the  enzyme  during  the 
reaction. 

Phenomena  of  a  similar  kind  are  known  in  pure  chemistry  and  are 
called  by  Ostwald  "  autocatalysis  "  (127,  ii.,  2,  p.  263).  When  an  ester 
is  acted  on  by  water  the  hydrolysis  is  at  first  very  slow,  but  as  acid  is 
set  free  the  reaction  is  rapidly  accelerated  as  the  acid  concentration  in- 
creases. This  is  positive  autocatalysis.  Other  cases  are  known  where 
the  catalyst  disappears  during  the  reaction,  as  in  the  transformation 
of  oxyacids  into  their  respective  lactones,  with  disappearance  of  the 
hydrion  which  was  acting  as  catalyst  (84).  Such  a  condition  is  nega- 
tive autocatalysis. 

Properly  speaking,  the  state  of  affairs  with  enzymes  is  not  quite  the 
same  as  this  autocatalysis,  where  there  is  production  or  disappearance 
of  a  body  which  acts  of  itself  as  a  catalyst.  Enzymes  produce  bodies 
which  are  not  necessarily  themselves  catalysts  for  the  reaction,  but  which 
act  by  increasing  or  decreasing  the  power  of  the  enzyme  itself.  The 
phenomena  are  sufficiently  alike  to  make  it  a  matter  of  convenience  to 
use  the  same  name. 

The  various  factors  affecting  the  rate  of  enzyme-action  dealt  with  up 
to  the  present  may  now  be  summed  up  as  follows  : — 

^     Causes  of  retardation  : — 

1.  Reversibility. 

2.  Combination  of  enzyme  with  products. 

3.  Negative  autocatalysis.     This,  with  the  previous  factor,  leads 

to  reversible  inactivation  of  the  enzyme. 

4.  Destruction  or  similar  drastic  change  in  the  properties  of  the 

enzyme,  irreversible  inactivation. 

«—    Causes  of  acceleration  : — 

1.  Combination  between  enzyme  and  substrate  when  the  latter 

is  in  relatively  large  excess.  This  is  strictly  to  be  regarded 
rather  as  a  retardation  of  the  early  stage,  leading  to  a  linear 
portion  of  the  time-curve. 

2.  Positive  autocatalysis. 

All  of  these,  with  the  exception  of  the  first,  reversibility,  are  included 
in  the  intensity-factor  of  Visser.  They  are  of  very  different  relative 
importance  in  connection  with  the  various  enzymes.  In  most  cases  the 
position  of  equilibrium  is  only  affected  by  the  reversibility-factor ;  the 

4 


50  THE  NATURE  OF  ENZYME-ACTION 

various  components  making  up  the  intensity-factor  do  not  cause  any 
change  in  this  equilibrium,  except  in  those  few  cases  where  it  appears 
that  the  enzyme  itself  enters  into  the  components  of  the  equilibrium. 
When  regarded  in  their  influence  on  the  velocity-constant,  as  calculated 
by  the  logarithmic  formula  of  the  simple  unimolecular  reaction,  the 
retarding  causes  produce  a  steady  fall  in  the  values,  while  the  accelerat- 
ing causes  produce  a  steady  rise,  as  in  the  exceptional  case  of  invertase. 

In  the  above  treatment  of  the  kinetics  of  enzyme-action,  it  has  been 
tacitly  assumed  that  the  reactions  take  place  in  a  homogeneous  system, 
viz.y  in  true  solution,  whereas  enzymes,  as  we  know,  are  colloids,  t.e., 
suspensions  of  ultra-microscopic  solid  particles,  so  that  the  systems  with 
which  we  have  to  do  are  heterogeneous.  It  has,  in  fact,  been  shown 
by  Dietz  (48,  p.  291)  that,  in  the  particular  case  investigated  by  him, 
the  reaction  takes  place  entirely  in  the  solid  enzyme  phase.  At  the 
same  time,  the  rate  of  diffusion  of  the  substrate  and  products  is  so  great, 
compared  to  the  rate  of  the  reaction  itself,  that  no  appreciable  error  is 
introduced  by  disregarding  the  diffusion-factor.  The  reaction,  in  this 
respect,  is  similar  to  that  investigated  by  Loevenherz  (113),  who  found 
that  when  various  esters  are  hydrolysed  by  hydrochloric  acid  in  a 
heterogeneous  system  of  water  and  benzene,  equilibrium  of  the  reacting 
bodies  is  very  rapidly  established  between  the  two  phases.  The  hydro- 
lysis takes  place  in  the  phase  in  which  the  velocity  is  the  greater,  in 
this  case  in  the  aqueous  hydrochloric  acid,  in  the  case  of  Hpase  in  the 
particles  of  the  enzyme.  Whether  this  applies  to  all  enzyme-actions 
cannot  be  stated  with  certainty  as  yet;  Arrhenius  (9,  p.  142),  however, 
makes  the  following  statement :  "  The  study  of  the  velocities  of  reactions 
in  heterogeneous  systems  indicates  that  they  behave  very  nearly  in  the 
same  manner  as  in  homogeneous  systems.  This  observation  has  often 
been  made  concerning  the  velocity  of  reactions  in  heterogeneous 
systems.  It  depends  on  the  circumstance  that  by  means  of  the  ex- 
perimental arrangements  the  diffusion  goes  on  so  rapidly  that  it  does 
not  perturb  the  chemical  processes.  If  capillary  tubes  are  employed 
this  cannot  be  said  to  be  the  case,  and  therefore  Mett's  tubes  should 
not  be  used  for  quantitative  measurements." 

Indirect  evidence  as  to  the  relatively  unimportant  part  played  by 
diffusion  is  afforded  by  the  temperature  coefficient  of  enzyme  reactions, 
which  is  unusually  high,  as  we  shall  see  in  the  next  section.  Diffusion, 
being  a  physical  process,  has  a  very  low  temperature  coefficient. 

It  may  serve  to  bring  together  the  facts  of  the  previous  pages  if  a 
brief  account  be  given  of  the  general  differential  equation  which  Benja- 
min Moore  (120,  p.  83)  has  put  forward  for  enzyme-actions.  We  will 
consider  that  we  are  dealing  with  a  reaction  which  is  unimolecular  in 
the  hydrolytic  direction  and  bimolecular  in  the  reverse  direction,  such 
as  invertase  on  cane-sugar  or  emulsin  on  salicin.  If  the  reaction  pro- 
ceeded to  completion  in  the  former  direction  and  there  were  no 
change  in  the  intensity  of  the  enzyme,  the  velocity  of  the  reaction 
would  be  simply 

—  -  KT 

where  x  is  the  concentration  of  the  products  at  the  time  /,  K  is  the  velo- 
city-constant and  a  is  the  initial  concentration  of  the  substrate,  so  that 
a-x\s  the  concentration  of  the  substrate  at  the  time  t. 


THE  VELOCITY  OF  REACTION  51 

We  have  seen,  however,  that  the  reverse  reaction  cannot  always  be 
neglected,  so  that  we  must  provide  for  it  in  any  complete  formula.  In 
the  case  before  us  it  is  supposed  to  be  bimolecular,  and  its  velocity  will 
be  proportional  to  the  products  multiplied  together.  Since  they  are 
present  in  equal  amount,  instead  of  using  a  separate  symbol  for  each, 
we  may  put  the  square  of  one,  say  ^r2,  and  the  velocity-constant  of  this 
reaction  will  not  be  the  same  as  the  previous  one,  so  that  the  equation 
becomes 


where  Kx  is  the  constant  of  the  hydrolytic  process  and  K2  that  of  the 
reverse  process. 

Further,  some  factor  must  be  introduced  to  allow  for  the  change  in 
intensity,  positive  or  negative,  which  the  enzyme  undergoes  in  propor- 
tion to  the  amount  of  change  that  has  taken  place,  z>.,  in  mathematical 
language,  this  factor  is  some  function  of  the  concentration  of  the  sub- 
strate, which  is  a  -  x  for  the  first  member  and  x  for  the  second.  Using 
e  as  expressing  the  activity  of  the  enzyme,  we  may  assume  as  an  ap- 
proximation the  factor  introduced  by  Henri,  in  which  the  enzyme 

x 
value  is  multiplied  by  a  quantity  — ,  representing  the  stage  at  which  the 

reaction  has  arrived  in  the  one  direction  and  by  a~x  for   the  reverse 

a 

reaction.  The  original  a -x  must,  therefore,  be  increased  or  diminished 
by  e  —  (a  -  x\  and  similarly  for  the  reverse  member.  The  full  equation 
then  becomes 

•w 

X 


dt 

As  Moore  himself  says :  "  This  formula  is  too  complicated  for  applica- 
tion to  experimental  results  on  integration,  but  it  includes  all  the  ob- 
served experimental  cases,  that  is,  it  shows  a  stage  when  x  is  small 
where  the  reaction  is  linear,  a  stage  where  the  reaction  is  more  rapid 
than  the  simple  logarithmic  law  demands,  as  in  Henri's  experiments,  a 
stage  showing  a  falling  off  from  the  logarithmic  values,  as  in  the  later 
stages  of  Armstrong  and  of  Bayliss,  a  zero  stage  at  the  equilibrium- 
point,  a  reversed  velocity,  which  also  at  the  very  end  tends  to  become 
linear". 

It  would  lead  us  too  far  to  test  all  the  various  possibilities  of  this 
equation,  and  the  reader  must  be  referred  to  the  work  of  Moore. 
Suffice  it  to  say  that  practically  many  simplifications  can  be  made,  ^, 
for  example,  can  be  taken  as  unity  without  any  perceptible  error.  In 
any  case  it  is  a  matter  of  some  satisfaction  that  reactions  which  at  first 
sight  are  so  complex  as  those  in  which  enzymes  play  a  part,  can  be  ex- 
pressed in  a  mathematical  formula  which  is  not  merely  empirical,  but 
in  which  the  several  factors  have  a  definite  experimental  foundation. 

We  now  turn  our  attention  to  certain  factors  influencing  the  velo- 
city of  enzyme-action  which  are  capable  of  definite  experimental  modi- 
fication. Such  are  temperature,  initial  concentrations  of  substrate  and 

4* 


52  THE  NATURE  OF  ENZYME-ACTION 

of  enzyme,  and  addition  of  various  foreign  substances  such  as  electro- 
lytes or  antiseptics. 

Temperature. — As  a  general  rule  chemical  reactions  are  increased 
by  rise  of  temperature  in  a  way  that  has  been  formulated  by  van't 
Hoff  into  the  well-known  rule  that  for  every  rise  of  10°  the  rate  of  a 
reaction  is  about  doubled  or  trebled ;  that  is,  if  a  reaction  has  a  rate 
represented  by  2  at  10°,  it  will  become  4  at  20°,  8  at  30°  and  so  on. 
Put  into  the  form  of  a  curve,  this  will  rise  slowly  at  first  and  then 
with  increasing  steepness  until  it  rapidly  becomes  nearly  vertical. 

Enzymes  are  no  exception  to  this  rule,  indeed  the  temperature 
coefficient  for  this  class  of  bodies  is  frequently  high.  Tammann  found 
for  emulsin  between  60°  and  70°  a  value  of  7' 14,  I  found  for  trypsin 
between  20°  and  30°  a  value  of  5*3,  that  is,  it  took  5*3  times  as  long  to 
effect  the  same  amount  of  change  at  20°  as  at  30°. 

This  behaviour  holds  only  up  to  a  certain  temperature,  which  varies 
according  to  conditions;  up  to  this  point  raising  the  temperature  in- 
creases the  rate  of  change,  but  a  further  rise  slows  the  reaction  again. 
This  is  the  phenomenon  known  as  the  "optimum  temperature".  Since 
the  property  is  sometimes  regarded  as  a  mysterious  one  and  not  shared 
by  inorganic  catalysts,  it  is  necessary  to  examine  into  its  meaning. 

Some  suggestion  as  to  the  explanation  is  afforded  by  the  experi- 
ments of  Ernst  (57,  pp.  476-77)  on  the  action  of  Bredig's  colloidal 
platinum  on  a  mixture  of  oxygen  and  hydrogen  gases.  This  catalytic 
reaction  shows  a  temperature  optimum  precisely  similar  to  that  of 
enzymes.  The  property  common  to  both  being  the  colloidal  condition, 
it  is  natural  to  suspect  that  this,  with  its  sensitiveness  to  heat,  is  the  cause 
of  the  phenomenon  in  question. 

An  important  series  of  experiments  have  been  made  by  Frost 
Blackman  and  Miss  Matthaei  (29)  on  the  carbon  assimilation  of  the 
green  leaf,  which  give  a  complete  explanation  of  the  question  at  issue. 
It  is  to  be  admitted  that  the  chlorophyll  function  is  only  in  part  an 
enzyme-action,  but  the  phenomena  are  so  much  alike  that  there  can 
be  no  reasonable  doubt  that  what  applies  to  the  one  applies  to  the 
other  also.  The  activity  of  the  process  is  retarded  by  the  injurious 
effect  of  temperatures  above  a  certain  height,  and  this  by  some  kind  of 
coagulating  action  on  the  colloidal  bodies  responsible  for  the  reaction. 
Of  what  particular  nature  this  destructive  action  is  does  not  affect  the 
question — the  important  point  being  that  before  complete  abolition  the 
process  is  more  or  less  gradually  injured.  Here  then  comes  in  the  im- 
portance of  the  "  time-factor,"  on  which  Blackman  lays  much  stress,  and 
no  doubt  correctly.  Sachs  (141,  p.  116)  clearly  pointed  out  that  the 
higher  the  temperature  the  more  quickly  a  fatal  effect  ensued,  and  that 
short  exposure  to  a  very  high  temperature  may  not  kill,  while  a  pro- 
longed exposure  to  a  slightly  lower  temperature  was  fatal.  Now  the 
facts  shown  in  the  work  on  carbon  dioxide  assimilation  referred  to  above 
are  summarised  by  Blackman  as  follows:  — 

(1)  At  high  temperatures  (30°  and  above  for  the  leaves  of  cherry- 
laurel)  the  initial  rate  of  assimilation  cannot  be  maintained,  but  falls  off 
regularly. 

(2)  The  higher  the  temperature  the  more  rapid  is  the  rate  of  falling  off. 

(3)  The  falling  off  at  any  given  temperature  is  fastest  at  first  and 
subsequently  becomes  less  rapid. 


THE  VELOCITY  OF  REACTION 


53 


This  falling  off  makes  it  experimentally  impossible  to  determine 
the  highest  value  at  any  given  temperature,  since  it  is  obviously  necessary 
to  allow  the  reaction  to  continue  for  a  certain  time  in  order  to  obtain 


10 


20 


30 


40^ 


50^ 


FIG.  5. 


.....    60  70 

(From  Blackman.) 

sufficient  change  to  measure  it  with  any  accuracy.  We  can,  however, 
arrive  at  this  indirectly  by  forming  what  may  be  called  the  van't  Hofjf 
curve  on  the  basis  of  measurements  at  lower  temperatures.  Below  25° 


54  THE  NATURE  OF  ENZYME-ACTION 

the  rate  of  assimilation  does  not  fall  off  with  successive  estimations,  so 
that  by  estimations  at  temperatures  differing  by  10°  we  can  determine 
the  coefficient  for  10°.  In  the  case  of  the  cherry-laurel  this  is  2*1.  The 
dotted  curve  in  Fig.  5  (from  Black  man)  gives  the  calculated  initial 
values  for  higher  temperatures. 

Prolonged  estimations  were  then  made  at  higher  temperatures,  viz., 
3°'5>  37'5  an<3  40*5,  the  rate  of  falling  off  in  each  case  being  determined. 
"  To  plot  these  on  the  diagram  we  regard  the  base  line  as  having  only  a 
time  significance,  each  division  representing  two  hours,  and  plot  out  the 
falling  series  of  readings,  obtained  at  the  temperatures  mentioned,  in 
curves  starting  from  the  initial  values  indicated  on  the  theoretical  van't 
Hoff  curve." 

It  then  becomes  at  once  obvious  that  the  calculated  initial  value  and 
the  observed  subsequent  values  fall  into  one  fairly  harmonious  curve  for 
each  temperature.  "  We  thus  attain  a  graphic  demonstration  that  both 
methods  indicate  practically  identical  initial  values,  and  this "  "  affords 
satisfactory  evidence  that  such  values  actually  occur,"  though  they  last 
too  short  a  time  to  be  measured.  At  45°  there  is  a  still  more  rapid  fall 
of  assimilation,  for  which  no  suitable  data  are  available,  the  decline  to 
zero  taking  place  in  a  very  short  time.  This  is  the  curve  starting  from 
F.  "  Finally,  to  conclude  the  series  we  ought  to  find  a  temperature  at 
which  the  earliest  estimation  that  could  be  actually  made  would  give  no 
measurable  assimilation.  The  lowest  temperature  to  give  this  result 
might  be  called  the  '  extinction  temperature/  and  here  we  should  hy- 
pothecate that,  for  the  first  few  seconds  after  attaining  it,  each  chloro- 
plast  would  give  a  higher  assimilation  rate  than  at  any  lower  temperature, 
but  that  the  rate  would  immediately  fall,  and  that  so  rapidly  that  it 
would  become  nil  almost  at  once  (say  in  100  seconds,  for  the  accepted 
specific  extinction  temperature  would  of  course  have  to  be  arbitrarily 
defined  in  time-units)."  This  is  placed  at  48°  in  the  figure,  the  curve 
falling  vertically  from  G. 

Two  things  follow  from  the  above  results.  In  the  first  place  the 
apparent  optimum  temperature  will  vary  considerably  according  to  the 
time  which  has  elapsed  between  the  beginning  of  the  exposure  to  a 
particular  temperature  and  the  period  during  which  the  estimation  is 
made.  Secondly,  the  so-called  optimum  temperature  is  merely  an  ex- 
pression of  the  fact  that  at  a  certain  temperature  the  increased  velocity 
due  to  this  raised  temperature  is  more  than  sufficient,  for  a  time  only, 
to  counteract  the  rapid  destruction  of  the  enzyme.  It  has  therefore  a 
negligible  importance,  both  theoretically  and  practically. 

As  regards  this  extinction  temperature,  it  is  to  be  noted  that  various 
enzymes  differ  in  their  sensitiveness  to  a  raised  temperature.  It  has 
been  already  mentioned  that  it  was  found  by  Fraenkel  and  Hamburg 
that  the  "  diastase  "  prepared  from  malt  seemed  to  consist  of  two  enzymes, 
as  was  suggested  by  Duclaux  some  years  ago.  One  of  these,  called  by 
Duclaux  (52,  p.  392)  amylase,  acts  upon  starch  only  to  such  a  degree  as 
to  convert  it  into  dextrin  ;  the  other,  dextrinase,  is  capable  of  hydrolys- 
ing  this  dextrin  to  maltose.  From  the  researches  of  Brown  and  Heron 
(40)  and  of  Kjeldahl  (98)  it  appears  that  the  dextrinase  is  more  injured 
by  a  temperature  of  68°  than  the  amylase  is.  At  least  this  seems  to  be 
the  probable  explanation  of  the  fact  that  when  starch  paste  is  acted  on 
by  "  diastase  "  which  has  been  exposed  to  a  temperature  of  68°  there  is 


THE  VELOCITY  OF  REACTION  55 

less  maltose  and  more  dextrin  formed  than  when  the  enzyme  has  not 
been  so  heated.  Reference  may  also  be  made  to  the  interesting  fact 
that,  in  the  action  of  diastase  on  starch,  the  reaction  ends  when  the 
composition  of  the  products  is  80*8  per  cent,  maltose  and  19*2  per  cent, 
dextrin.  This  appears  to  be  due  to  a  reverse  conversion  of  maltose  into 
dextrin,  for,  if  the  mixture  be  subjected  to  the  action  of  a  mixture  of 
diastase  and  yeast,  although  the  yeast  is  only  able  to  ferment  the  mal- 
tose, the  dextrin  is  found  to  disappear  also  (38,  p.  105).  The  reason 
is  that,  as  the  maltose  is  removed  by  fermentation,  the  diastase  converts 
a  further  quantity  of  dextrin  into  maltose,  which  in  its  turn  is  attacked 
by  the  maltase  of  the  yeast  and  then  fermented. 

~^jj  Concentration  of  Substrate. — There  is  little  to  be  added  on  this  point 
to  what  has  been  already  stated.  The  rate  of  hydrolysis,  by  mass- 
action,  should  be  directly  proportional  to  the  concentration,  but  this 
actually  happens  only  when  the  concentration  of  the  enzyme  is  not  too 
low  with  respect  to  that  of  the  substrate.  When  the  latter  is  in  con- 
siderable excess,  the  "  combination  "  of  the  two  bodies  produces  a  state 
of  affairs  such  that  the  amount  of  change  is  nearly  equal  with  varying 
concentration  of  the  substrate,  as  is  shown  in  the  table  from  Frankland 
Armstrong  previously  quoted.  /In  some  cases,  as  in  that  of  the  action  of 
trypsin  on  gelatin,  the  rate  of  hydrolysis  is  actually  less  in  the  stronger 
solutions  as  shown  in  the  following  table,  which  gives  in  the  second 
column  the  change  of  electrical  conductivity  in  twenty-five  minutes 
in  solutions  of  gelatin  of  the  strengths  given  in  the  first  column : — 

Per  cent.  Gemmhos. 

10  ,         ,!        .         .         .         .     130 

8  .         .         ....     170 

4 240 

2 280 

This  effect  is  perhaps  due  to  some  obscure  influence  of  viscosity.  It  is 
not  so  marked  in  the  case  of  caseinogen,  in  which  I  found  the  rates  of 
change  in  the  first  stages  of  the  reaction  to  be  equal  for  concentrations 
of  8  per  cent.,  5  per  cent,  and  4  per  cent.,  while  that  for  10  per  cent, 
was  somewhat  less  than  that  of  8  per  cent. 

^  Concentration  of  Enzyme. — This  is  a  question  of  some  importance, 
since  certain  results  obtained  from  limited  observations  have  been  made 
the  basis  of  estimations  of  the  relative  amounts  of  enzyme  present  in 
solutions. 

With  regard  to  the  inorganic  catalysts,  it  is  usually  found  that  the 
velocity  of  the  reaction  is  in  direct  linear  proportion  to  the  amount  of 
the  catalyst  added.  This  is  not,  however,  a  universal  rule. 

Various  conflicting  statements  have  been  made  as  to  the  law  in  the 
case  of  enzymes.  Some  observers  have  found  a  direct  linear  propor- 
tion, others  that  high  concentrations  are  relatively  less  active  than 
lower.  Schiitz  (144)  and  Borissow  have  gone  so  far  as  to  formulate  a 
law  according  to  which  the  action  is  proportional  to  the  square  root  of 
the  concentration. 

It  will  be  clear,  from  what  has  been  stated  in  the"  previous  pages, 
that  these  discrepant  rules  may  all  be  correct,  but  that  they  apply  to 
different  relative  concentrations  of  enzyme  and  substrate,  or,  in  other 
words,  to  different  stages  of  the  reaction,  when  this  begins  with  relative 
excess  of  substrate. 


56  THE  NATURE  OF  ENZYME-ACTION 

When  the  enzyme  is  in  considerably  smaller  concentration  than  the 
substrate,  owing  to  the  whole  of  it  being  able  to  enter  into  effective 
combination  with  substrate,  the  velocity  of  the  reaction  is  in  direct 
linear  proportion  to  the  quantity  of  enzyme  present.  As  the  con- 
centration of  the  substrate  diminishes,  another  law  begins  to  make  its 
appearance,  so  that  the  greater  quantities  of  enzyme  have  relatively  less 
effect.  The  so-called  "  law  "  of  Schiitz  and  Borissow  is  one  particular 
case  of  this  relationship.  What  the  ratio  actually  is  varies  somewhat 
according  to  circumstances,  but  is  generally  some  root  less  than  the 
square-root.  The  probable  meaning  of  this  exponential  formula  will 
be  discussed  in  the  next  chapter.  In  an  actual  case  in  which  the  ques- 
tion was  investigated,  viz.,  in  the  action  of  trypsin  on  caseinogen,  the 
amounts  of  the  trypsin  varied  in  relative  amount  from  0*5  to  4.  The 
curves  of  Fig.  I  (p.  10)  were  drawn  from  a  part  of  this  experiment,  and 
a  glance  at  the  slope  of  the  several  curves  will  show  that  the  relative 
activity  of  the  different  concentrations  of  the  enzyme  is  quite  different 
at  the  different  stages.  For  instance,  between  twenty  and  forty 
minutes  after  the  commencement  of  the  reaction  the  slope  (  =  velocity 
of  change)  is  clearly  steeper  in  curve  2  than  in  curve  0*5,  whereas  be- 
tween 220  and  240  minutes  the  reverse  is  the  case.  It  will  be  seen 
also  that  at  the  same  period  of  220  to  240  minutes  the  curves  of  4,  2*5 
and  2  have  the  same  slope,  so  that  it  might  appear  that  the  enzyme- 
concentration  was  the  same  in  all  three. 

If  we  consider  the  numerical  data  from  which  the  curves  were  con- 
structed, the  reaction  is  seen  to  come  practically  to  a  standstill  when 
the  electrical  conductivity  had  risen  by  2,200  gemmhos,  so  that  a 
change  of  800  gemmhos  may  be  taken  as  representing  about  one-third 
of  the  total.  In  the  first  table  below,  the  first  column  gives  the  relative 
trypsin -con  tent,  the  second  the  number  of  minutes  from  the  com- 
mencement of  the  reaction  until  a  change  of  800  gemmhos  had  been 
attained,  while  the  third  gives  the  calculated  times  on  the  assumption 
of  a  direct  linear  relationship : — 

Trypsin-content.  Time  Observed.  Time  Calculated. 

4  4'5  5 
2-5                                           7-5  8 

2  IO  10 

I  IQ  20 

o'5  37  40 

The  substrate  here  was  ammonium  caseinogenate,  in  2*5  per  cent,  solu- 
tion. The  following  is  a  similar  experiment  with  gelatin  in  5  per  cent, 
solution  : — 

10  2-5  2-67 

5  4*8  5'3 
2-5                                           5-9                                          10-6 

I  267  26-7 

0'5  5i  53H 

0-25  90  106-8 

The  value  underlined  is  obviously  an  error  of  experiment.  The  linear 
relationship  in  this  early  part  of  the  reaction  is  sufficiently  unmistak- 
able. 

The  next  table  gives  another  series  of  data  from  the  caseinogen  ex- 
periment, but  at  a  later  stage  in  the  reaction,  viz.,  the  times  taken  by 


THE  VELOCITY  OF  REACTION 


57 


the  various  concentrations  of  enzyme  to  effect  a  change  from  1,300  to 
i,  800  gemmhos  :  — 


V 

Relative 
Trypsin- 

Time  Taken 

Mean  Velocity  = 

Specific  Activity  =—  . 

content 

(/). 

-  x  looo  -  V. 

(c). 

t 

Found. 

Calculated  by  Square- 
root  Law. 

4 

4i 

24 

6 

(6) 

48 

20-8 

8-3 

7'6 

2 

55 

18-2 

g-i 

9'3 

I 

Si 

12-4 

12-4 

127 

0-5 

144 

7 

14 

17-5 

A  glance  at  the  numbers  in  the  second  column  shows  that  the  linear 
relationship  no  longer  holds  ;  in  fact,  if  the  mean  velocity  during  the 
period  be  calculated,  the  numbers  of  the  third  column,  which  show  that 
the  smaller  concentrations  are  relatively  more  active  than  the  larger, 
are  obtained.  The  fact  is  brought  out  more  definitely  if  we  take  the 
specific  activities,  i.e.,  the  activity  per  unit  amount  of  enzyme,  as  is  done 
in  the  fourth  column.  In  the  last  column  are  given  the  values  given  by 
the  Schutz-Borissow  law,  which  in  this  stage  of  the  reaction  gives  fairly 
good  results. 

The  general  conclusion  to  be  drawn  from  these  data  is  that  neither 
the  linear  nor  the  exponential  law  can  be  practically  applied  except 
when  direct  experiments  with  corresponding  relative  concentrations  of 
enzyme  and  substrate  have  shown  what  law  holds.  The  fact  that 
different  observers  have  formulated  different  laws  is  to  be  explained;  by 
their  having  worked  with  different  relative  concentrations  of  enzyme 
and  substrate  or  at  different  stages  in  the  reaction,  which  both  practi- 
cally come  to  the  same  thing. 

In  the  case  of  trypsin  given  above,  if  we  had  taken  as  our  basis  of 
comparison  the  times  taken  to  effect  a  change  from  the  commencement 
to  i, 800  gemmhos,  it  is  plain  that  the  law  would  have  been  found  to  be 
between  the  linear  and  the  square-root  laws.  A  series  of  measurements 
taken  with  gelatin  showed  that  the  ratio  was  about  the  i'5th  root,  instead 
of  the  square-root. 

The  significance  of  the  exponential  law  will  be  discussed  in  the  next 
chapter. 

^  Action  of  Electrolytes. — Enzymes  being  colloids  are  very  sensitive  to 
the  action  of  electrolytes.  In  some  cases  indeed  they  may  be  said  to 
be  inactive  in  the  absence  of  some  one  or  other  of  these  agents  ;  pepsin 
is  practically  inactive  without  the  presence  of  hydrion,  trypsin  without 
hydroxidion.  In  these  cases,  therefore,  the  electrolyte  plays  the  part 
of  a  body  which  we  shall  learn  later  to  call  a  co-enzyme. 

The  action  of  neutral  salts  will  best  be  discussed  in  the  next  chapter, 
since  it  seems  to  be  connected  with  the  facilitation  or  otherwise  of 
approximation  of  enzyme  and  substrate  to  one  another,  at  least  in  so 
fai  as  our  present  knowledge  allows  us  to  form  any  general  view  on  the 
question. 


58  THE  NATURE  OF  ENZYME-ACTION 

In  certain  cases  bodies  play  the  part  of  specific  activators,  e.g., 
asparagine  on  amylase  (54,  p.  143). 

*•—  Antiseptics. — A  detailed  study  of  this  part  of  our  subject  does  not 
enter  into  the  scope  of  the  present  book,  but  on  account  of  its  practical 
value  in  enabling  the  distinction  to  be  made  between  the  results  of  the 
activity  of  living  cells  and  that  of  enzymes  a  few  words  are  required. 
Emil  Fischer  has  recommended  the  use  of  toluene  as  most  appropriate 
for  this  purpose  (63).  It  is  chemically  inert  and  has  scarcely  any 
destructive  action  on  enzymes,  while  it  prevents  the  growth  of  proto- 
plasmic structures  and  therefore  excludes  phenomena  dependent  on  this. 
How  far  portions  of  cell-protoplasm  can  be  said  to  be  "  killed  "  by  it  is 
another  question,  which  is  perhaps,  at  bottom,  an  idle  one,  since  there 
are  in  all  probability  numerous  stages  of  complexity  between  an  enzyme 
like  invertase  or  lipase  and  actual  portions  of  cells,  such  a  stage  being 
zymase.  At  the  same  time,  so  far  as  a  body  exerts  catalytic  actions, 
it  is  found  that  it  obeys  ordinary  chemical  laws,  so  that  we  are  justified 
in  treating  it  as  an  enzyme. 

When  the  phenomena  due  to  growth  make  their  appearance  other 
laws  must  be  taken  into  consideration. 

For  a  detailed  investigation  of  the  action  of  various  antiseptics  on 
trypsin  the  reader  is  referred  to  the  work  of  Kaufmann  (96). 

It  is  important  to  remember  that  a  particular  antiseptic  may  be 
comparatively  harmless  to  one  enzyme  and  yet  very  injurious  to  another. 
It  is  impossible  in  the  present  state  of  our  knowledge  to  give  any  ex- 
planation of  this  fact. 


CHAPTER  VII. 

THE  NATURE  OF  THE  COMBINATION  BETWEEN  ENZYME  AND 

SUBSTRATE. 

ABUNDANT  evidence  has  been  given  to  show  that,  in  order  that  an 
enzyme  may  exert  its  activity,  a  preliminary  combination  of  some  kind 
between  it  and  the  substrate  is  necessary.  Now  there  are  many  pheno- 
mena, which  we  have  met  with  in  the  preceding  pages,  that  assist  us  in 
forming  a  conclusion  as  to  the  nature  of  this  combination. 
x»  As  colloids,  enzymes  will  be  particularly  prone  to  form  what  we 
have  called  "adsorption-compounds"  (15).  Since  doubt  has  recently 
been  expressed  by  Brailsford  Robertson  (139)  as  to  the  actual  existence 
of  phenomena  of  this  kind,1  it  is  well  to  repeat  what  is  meant  by  the 
use  of  the  name  in  the  present  work.  There  can  be  no  doubt  that  there 
is  a  condensation  of  dissolved  bodies  at  the  interface  between  the  sol- 
vent and  the  solids  suspended  in  it  (Willard  Gibbs,  189).  Moreover,  the 
degree  of  this  condensation  is  frequently  in  unmistakable  relation  with 
the  chemical  configuration  of  the  bodies  concerned.  How  far  it  is 
justifiable  in  the  present  state  of  our  knowledge  to  continue  in  imagina- 
tion the  process  until  molecular  dimensions  are  reached  is  another 
question.  In  certain  cases  also  phenomena  due  to  "  solid  solution," 
with  slow  diffusion  in  the  solid  phase,  add  themselves  on  and  assist  in 
the  production  of  the  exponential  law  of  formation  of  these  adsorption- 
compounds.  Such  a  case  is  that  treated  by  Travers  (156),  viz.,  the 
"  occlusion  "  of  gases  by  charcoal. 

Davis,  also  (181),  has  conclusively  shown  that,  in  the  adsorption  of 
iodine  by  carbon,  there  are  two  factors,  a  surface  absorption,  which 
takes  place  rapidly,  and  a  "  solid  solution "  in,  or  diffusion  into,  the 
substance  of,  the  carbon  (/absorption).  This  latter  process  is  slow  and 
arrives  at  a  state  of  equilibrium  only  after  many  days. 

The  distinguishing  characteristic  is  just  the  form  of  the  law  which 
expresses  the  relation  of  the  composition  of  the  product  to  the  con- 
centration of  the  bodies  forming  it ;  for  example,  in  a  concrete  case 
the  amount  of  dye  taken  up  by  a  stuff  is  not  in  direct  linear  relation  to 
the  concentration  of  the  dye,  but  is  relatively  greater  the  lower  this  con- 
centration is. 

If  we  refer  back  to  the  table  on  p.  57  giving  the  times  taken  by  differ- 
ent concentrations  of  trypsin  to  effect  a  change  from  1,300  to  1,800  gemm- 
hos,  it  will  be  seen  that  the  lower  concentrations  are,  weight  for  weight, 
more  active  than  the  higher  ones.  If  we  make  the  reasonable  assumption 
that  the  rate  of  change  is  in  proportion  to  the  amount  of  "  compound  " 
of  enzyme  and  substrate  in  existence  at  the  time,  it  will  be  seen  that 
the  result  is  what  would  be  expected  if  this  combination  were  of  the 

1  See  also  the  criticism  of  Bratlsford  Robertson's  view  by  van  Slyke  (182). 

59 


6o  THE  NATURE  OF  ENZYME-ACTION 

nature  of  an  adsorption-compound,  since  more  trypsin  will  be  in  associa- 
tion with  the  substrate  in  proportion  to  the  concentration  of  the  enzyme 
when  the  latter  is  lower.  The  conditions  in  the  first,  linear,  stage  are 
not  inconsistent  with  this  interpretation ;  in  the  case  of  congo-red  and 
paper,  when  the  amount  of  the  dye  is  very  small  compared  with  that 
of  the  paper,  practically  all  of  the  dye  is  taken  up  by  the  paper,  since 
it  is  necessary  to  examine  the  solution  in  a  deep  layer  in  order  to  detect 
any  colour  in  it ;  such  an  amount  would  be  within  the  errors  of 
experiment  in  enzyme  work. 

The  exponential  law  of  activity  of  enzymes  in  relation  to  concentra- 
tion, of  which  the  square-root  law  is  a  particular  case,  is  the  mathe- 
matical expression  of  the  above  facts. 

That  this  state  of  affairs  is  due  to  the  colloidal  nature  of  enzymes, 
and  therefore  an  adsorption  phenomenon,  is  indicated  by  the  fact  pointed 
out  by  Bredig  and  M.  v.  Berneck  (35,  p.  317)  that  in  the  catalysis  of 
hydrogen  peroxide  by  colloidal  metals  the  same  kind  of  law  holds, 
contrary  to  what  obtains  in  the  inversion  of  cane-sugar  by  hydrion, 
where  the  law  of  linear  proportionality  holds. 

In  the  experiments  of  Dietz  (48,  p.  314),  to  which  frequent  reference 
has  been  made,  it  was  found  that  the  velocities  of  the  reactions  were 
proportional  to  the  square-roots  of  the  substrate-concentration.  A 
little  consideration  will  show  that  this  is  another  aspect  of  the  same  ex- 
ponential relationship  dealt  with  above.  It  was  shown  also  directly  that 
adsorption  did  actually  occur. 

Another  fact  of  importance  was  made  out  in  these  experiments  with 
lipase,  showing  that  the  adsorption-compound  of  enzyme  and  substrate 
is  the  active  system.  As  the  enzyme  was  in  the  form  of  minute  particles 
insoluble  in  the  solution  of  substrate,  the  system  was  heterogeneous,  and 
it  was  shown  in  the  following  way  that  the  reaction  took  place  entirely 
in  the  solid  phase : — 

A  mixture  of  amyl  alcohol  and  butyric  acid  was  acted  on  by  the 
enzyme  preparation.  The  initial  value  of  the  acid  was  equivalent  to 
6*50  c.c.  of  barium  hydroxide  solution.  After  two  hours  forty-six  min- 
utes the  value  was  5*28  c.c.  From  this  mixture  20  c.c.  were  now  re- 
moved, filtered,  and  the  clear  filtrate,  now  free  from  enzyme,  put  again 
into  the  thermostat  After  a  further  twenty-three  hours  forty-six  min- 
utes the  original  solution  had  arrived  at  a  value  of  1 79  c.c.  barium  hy- 
droxide, while  the  filtered  solution  remained  constant  at  5*30  c.c. 

According  to  the  author  the  process  probably  takes  place  in  the 
following  manner  :  "  The  bodies  in  the  solution  diffuse  into  the  ferment- 
phase  and  are  here  caused  to  interact  by  the  agency  of  the  enzyme. 
The  bodies  produced  in  their  turn  diffuse  out  into  the  solution." 

A  remarkable  fact,  which  resulted  from  the  experiments  of  Dietz, 
appears  to  have  its  explanation  in  adsorption  processes  of  some  kind. 
It  has  been  already  made  sufficiently  clear  that  the  equilibrium  arrived 
at  under  the  action  of  lipase  is  a  real  one.  The  same  point  is  reached 
from  both  sides  and  it  is  independent  of  the  amount  of  catalyst.  It  is 
therefore  somewhat  unexpected  to  find  that,  when  acids  are  used  as 
catalysts,  the  equilibrium  is  not  the  same  as  that  with  the  enzyme. 
Under  similar  conditions,  in  the  former  case,  i.e.,  in  homogeneous 
system,  the  equilibrium  is  reached  with  85*5  per  cent,  of  ester,  while  in 
the  case  of  the  enzyme  the  value  reached  is  only  75  per  cent.  As 


COMBINATION  OF   ENZYME  AND  SUBSTRATE      61 

pointed  out  by  Dietz,  this  circumstance  appears  to  present  an  opportunity 
of  evading  the  second  law  of  thermodynamics,  in  that  heat  can  be 
transformed  into  work  at  constant  temperature  by  merely  changing 
acid  for  enzyme  and  back  again  in  a  cycle.  But  this  is  impossible  unless 
energy  is  in  some  way  supplied  to  the  system  by  the  enzyme  itself,  in 
which  case  it  would  not  be  in  the  same  state  at  the  end  of  the  reaction 
as  at  the  beginning.  Experiments  made  for  the  purpose  of  detecting 
changes  in  the  enzyme  during  the  reaction  showed  that  there  were  none, 
so  that  some  obscure  surface-energy  change  must  in  all  probability  be 
the  cause  of  the  paradoxical  behaviour  (48,  p.  323). 

This  theory  (Bayliss  [15,  p.  300])  of  the  relative  proportion  of  the 
enzyme  combined  with  the  substrate,  compared  to  that  in  the  remainder 
of  the  system,  will  be  seen  to  resemble  closely  that  of  Victor  Henri 
(81),  although  this  investigator  does  not  specifically  refer  to  colloidal- 
complexes  or  adsorption.  It  also  has  possibly  some  relation  to  that  of 
Armstrong  (6  and  8)  as  to  the  association  of  enzyme  with  water  and 
sugar. 

It  must  be  clearly  understood  that  it  is  only  the  preliminary  com- 
bination of  enzyme  and  substrate  that  follows  the  law  of  adsorption. 
After  close  association  has  taken  place,  the  proper  chemical  actions, 
due  to  the  agency  of  the  enzyme,  begin  to  make  their  appearance. 
The  criticism  of  Brailsford  Robertson  (137,  p.  375),  that  adsorption,  as 
a  physical  process,  has  a  low  temperature  coefficient,  whereas  enzyme- 
action  as  a  whole  has  the  high  coefficient  of  a  true  chemical  reaction, 
is  therefore  beside  the  point. 

Objection  may  be  taken  to  the  view  here  put  forward,  on  the  ground 
that  no  account  seems  to  be  taken  of  the  very  specific  nature  of  certain 
enzymes.  There  is,  however,  considerable  reason  for  believing  that 
chemical  relationship  plays  an  important  part  in  adsorption  phenomena. 
The  "  lock-and-key "  simile  of  Fischer  may  be  taken  to  illustrate  this 
fact,  so  that  it  may  be  said  that  the  chemical  configuration  of  the  sur- 
faces of  contact,  or  the  molecular  shape  of  the  constituents  of  the 
surfaces,  are  potent  factors  in  determining  the  possibility  of  intimate 
contact  between  them.  Using  a  somewhat  gross  illustration,  a  surface 
formed  of  rounded  elevations,  or  projecting  spikes,  cannot  come  into 
close  contact  with  a  flat  one  (see  also  Starling  [150,  p.  40]). 

The  relation  of  enzymes  to  optically  isomeric  bodies  affords  sup- 
port to  the  view  of  shape  as  a  factor  in  enzyme-action.  This  may  be 
seen  in  Fig.  6,  which  represents  very  diagrammatically  the  relation  of 
the  glucosides  to  maltase  and  emulsin.  Since  bodies  of  three  dimen- 
sions cannot  be  readily  drawn  on  a  plane  surface,  the  postulate  must 
be  made  that  the  figures  are  unable  to  move  out  of  the  plane  of  the 
paper.  This  being  so,  it  will  readily  be  seen  that  maltase  can  enter 
into  intimate  contact  with  the  a-glucoside  but  not  with  the  /3-glucoside, 
whereas  emulsin,  assumed  to  be  the  mirror-image  of  maltase,  can  ap- 
proach closely  to  the  latter,  but  not  to  the  former. 

There  is,  moreover,  actual  evidence  of  a  certain  degree  of  specificity 
in  more  unquestionable  cases  of  adsorption.  It  was  shown  by  the  pre- 
sent author  (14,  p.  213)  that  gelatin  will  take  up  considerably  more 
acid-fuchsin  than  it  will  of  congo-red,  while  filter-paper  takes  up  the 
same  amount  of  both.  Gelatin  also  takes  up  calcium  salts  more  readily 
than  potassium  salts ;  the  same  holds  for  filter-paper,  as  shown  by 


62  THE  NATURE  OF  ENZYME-ACTION 

Schonbein  (143),  who  found  that,  when  strips  of  paper  were  immersed 
in  solutions  of  various  salts,  the  height  to  which  calcium  and  barium 
rose  was  less  than  that  to  which  potassium  rose,  although  the  height  to 
which  the  water  rose  was  the  same  in  each. 

Zunz  (186)  has  shown  that  while  some  of  the  proteoses  contained 
in  Witte's  peptone  are  precipitated  as  adsorption-compounds  with  mastic 
in  the  method  of  Michaelis  and  Rona  (116),  others  are  not.  It  can 
scarcely  be  held  that  chemical  combination  takes  place  between  mastic 
and  these  proteins. 

The  view  taken  in  this  monograph  as  to  the  existence  of  specific 
"adsorption-affinity"  is  contrary  to  that  of  Freundlich  (175),  who  states 
that  the  amount  of  adsorption  is  independent  of  the  nature  of  the  ad- 
sorbing agent  if  equal  surfaces  be  compared.  Davis  (181),  however, 
has  shown  that,  even  to  the  simple  case  of  carbon  and  iodine,  this  gen- 
eralisation does  not  apply.  In  his  experiments  it  was  found  that  the 
following  relative  amounts  of  iodine  were  left  unadsorbed,  under  the 
same  conditions : — 

By  cocoa-nut  charcoal         I  "236 

By  bone  charcoal  0*522 

By  sugar  charcoal  0799 


Maltaso.  Emulsin. 

FIG.  6. 

This  investigator  states  that  his  experiments  indicate  that  the  surface 
adsorption  is  specific,  while  the  diffusion-factor  is  independent  of  the 
nature  of  the  carbon.  This  fact  is  significant  in  connection  with  what 
has  been  said  above  as  to  the  effect  of  configuration  of  surface. 

In  the  case  of  the  enzymes  themselves,  a  definite  case  of  special 
adsorption-affinity  has  been  brought  forward  by  Hedin  (i  80).  Kieselguhr 
takes  up,  from  a  mixture  of  the  two  proteolytic  enzymes  of  the  spleen, 
large  quantities  of  the  a-protease,  leaving  the  y8-protease  almost  un- 
touched. Charcoal,  on  the  contrary,  adsorbs  the  same  proportion  of 
both  enzymes.  The  a-protease,  it  will  be  remembered,  acts  in  alkaline 
solution,  the  y3-protease  in  acid  solution ;  it  is  possible  that  the  two 
bodies  have  electrical  charges  of  opposite  sign  and  that  this  circum- 
stance may  play  a  part  in  the  phenomena  (Bayliss  [14,  p.  206]).  The 
results  of  Michaelis  (187)  on  invertase  are  also  to  the  point  in  this  con- 
nection. This  enzyme  is  adsorbed  by  certain  inert  powders,  while  being 
left  unadsorbed  by  others. 

Aggazzotti  (4)  has  recently  published  some  observations  on  the 
action  of  enzymes  as  seen  in  the  ultra-microscope.  These  results,  ac- 
cording to  the  author,  give  support  to  the  theory  of  combination  between 
enzyme  and  substrate,  although  it  must  be  admitted  that  their  interpre- 


COMBINATION  OF  ENZYME  AND  SUBSTRATE       63 

tation  is  not  easy.  It  will  be  sufficient  to  state  here  that,  at  the  end  of 
the  reaction,  certain  large  granules,  few  in  number,  are  left  unattacked. 
These  bodies,  which  are  considerably  larger  than  the  particles  of  the 
enzyme  or  substrate,  are  regarded  as  consisting  of  a  colloidal-complex 
of  enzyme  with  certain  products  of  the  reaction.  The  changes  which 
occur  on  first  adding  the  enzyme  to  the  substrate  are  apparently  too 
complex  to  enable  any  conclusions  to  be  drawn  whether  union  of  these 
two  bodies  took  place. 

The  solubilities  of  some  enzymes  indicate  a  relationship  to  their  re- 
spective substrates.  As  shown  by  Dietz  (48,  p.  286),  pancreatic  tissue 
can  be  washed  free  from  trypsin  by  water,  which  leaves  the  lipase  behind. 
Lipase  is,  however,  readily  soluble  in  glycerol,  and  has  been  stated  to 
be  soluble  in  ether,  or  rather  in  an  ethereal  extract  of  liver,  probably 
containing  lecithin  (Ramond). 

Reference  has  been  made  in  a  previous  chapter  to  the  effect  of 
electrolytes  on  adsorption.  If  the  enzyme  and  substrate  are  colloids 
and  both  with  an  electric  charge  of  the  same  sign,  it  is  plain  that  a 
certain  mutual  repulsion  will  tend  to  obstruct  the  formation  of  a  com- 
pound, just  as  in  my  experiments  negative  paper  takes  up  very  little 
negative  congo-red,  but  when  given  an  opposite  charge  by  a  kation,  a 
large  amount  of  the  dye  is  adsorbed.  It  seems  then  very  probable  that 
the  action  of  electrolytes  on  enzymes  may  in  some  cases  be  referable  to 
this  circumstance.  As  yet  the  question  awaits  investigation.  Trypsin 
is  stated  by  Victor  Henri  to  be  electro-negative l ;  in  agreement  with 
this  I  have  found  that  the  amount  of  it  which  is  taken  up  by  paper  is 
increased  by  the  presence  of  calcium  sulphate  (14,  p.  226).  It  is  also 
interesting  to  note  that  calcium  ions  have  been  shown  by  Pottevin  (136) 
and  by  Kanitz  (94)  to  increase  the  activity  of  both  trypsin  and  lipase ; 
their  action  is  therefore  not  specific,  but  is  probably  due  to  their  effect 
on  adsorption  of  substrate  by  enzyme.  This  effect  of  kations  on  trypsin 
is  not  obtained  except  in  very  low  concentrations  of  the  electrolytes ; 
in  higher  concentrations  they  are  injurious.  This  fact  is  again  in  com- 
plete agreement  with  the  facts  of  adsorption,  as  seen  in  the  case  of  congo- 
red  and  paper.  Here  also  if  the  concentration  of  the  calcium  is  greater 
than  about  0*005  molar,  the  dye  is  precipitated  in  such  a  way  that 
the  large  particles  are  not  taken  up  by  the  paper  at  all ;  the  colloid 
must  not  be  caused  to  agglomerate  or  the  adsorption  will  not  take 
place. 

On  the  whole  it  seems  evident  that  the  action  of  electrolytes  may 
be  due  to  very  various  causes  in  different  cases,  so  that  it  is  impossible 
to  formulate  statements  of  general  application.  In  the  next  chapter  it 
will  be  seen  that  some  enzymes  are  quite  inactive  without  the  presence 
of  electrolytes. 

Zymoids. — There  are  some  facts  which  give  support  to  Ehrlich's 
view  that  the  combining  power  and  fermentative  activity  are  functions 
of  distinct  "side-chains".  It  was  found  by  Korschun  (104),  when  in- 
vestigating the  relations  between  rennet  and  >its  anti-body,  that  by 
filtration  through  porous  clay  a  solution  of  rennet  could  be  separated 
into  several  fractions  which,  by  appropriate  dilution  of  the  stronger 
fractions,  could  be  brought  to  the  same  strength  as  regards  combina- 
tion with  the  anti-body,  but  which  differed  considerably  in  their  power 

1  See  Iscovesco  (91). 


64  THE  NATURE  OF  ENZYME-ACTION 

of  clotting  milk.  In  other  words,  the  original  solution  appeared  to 
contain  a  modified  form  of  the  enzyme  analogous  to  Ehrlich's  "  tox- 
oids " ;  that  is,  a  part  of  the  enzyme  had  lost  its  characteristic  action 
while  retaining  its  power  of  combining  with  the  anti-body.  I  have 
myself  (13,  p.  271)  met  with  some  facts  which  point  to  the  production 
of  a  similar  modification  of  trypsin  by  warming  to  25°  for  a  day  or  so. 
I  suggested  calling  these  modified  enzymes  "zymoids".  The  experi- 
ments of  Beam  and  Cramer  (21)  are  also  of  interest  in  this  connection. 


CHAPTER  VIII. 

CO-ENZYMES  AND  ANTI-ENZYMES. 

IT  was  noticed  by  Magnus  (115)  that,  when  an  extract  of  liver  was 
subjected  to  dialysis,  the  lipolytic  power  which  it  originally  possessed 
was  gradually  lost,  but  was  regained  when  the  dialysate  was  added. 
This  experiment  shows  that  what  may  be  called  the  lipoclastic  system 
of  the  liver  consists  of  more  than  one  component,  each  of  which  is 
separately  inactive.  The  inactive  dialysed  extract  prepared  by  Magnus 
was  also  restored  to  activity  by  the  addition  of  boiled  liver-extract,  or 
by  a  similar  extract  from  which  proteins  had  been  precipitated  by 
uranyl-acetate.  The  activating  body  was  soluble  in  alcohol,  but  not  in 
ether,  and  was  not  present  in  the  ash  of  liver.  The  component  which 
did  not  dialyse  was  destroyed  by  boiling  and  may  therefore  be  regarded 
in  a  sense  as  the  enzyme  proper,  while  the  dialysable,  thermostable,  body 
or  bodies  may  be  called  the  "  co-enzyme  ". 

This  name  "co-enzyme"  or  "co-ferment"  was  introduced  by  Ber- 
trand  (26)  to  express  the  great  increase  in  the  oxidising  power  of 
laccase  brought  about  by  the  addition  of  manganese  salts  in  minute 
quantity.  It  has  not,  however,  been  shown  that  laccase  is  actually  in- 
active apart  from  manganese,  so  that  the  original  use  of  the  name  "  co- 
enzyme  "  was  rather  in  the  sense  of  what  we  now  sometimes  call  an 
"accelerator,"  similar  to  asparagine  in  connection  with  amylase,  as 
already  mentioned.  Bertrand  also  applied  the  name  to  calcium  salts, 
in  the  case  in  which  they  are  apparently  necessary  for  the  action  of 
pectase  on  pectin. 

Harden  and  Young  (73)  have  described  a  case  of  well-marked  co- 
enzyme  relationship  in  the  alcoholic  enzyme  of  yeast-juice.  When 
yeast-juice,  prepared  by  Buchner's  method,  is  filtered  under  pressure 
through  a  Martin's  gelatin  filter,  the  colloids  are  left  behind  on  the 
gelatin.  The  substance  so  obtained,  which  would  be  expected  to  con- 
tain the  enzyme,  showed  itself  to  be  inactive,  but  when  mixed  with  a 
portion  of  the  filtrate,  which,  alone,  is  equally  inactive,  it  became 
capable  of  exciting  vigorous  fermentation.  The  co-enzyme  found  in 
the  filtrate  is  dialysable  and  not  destroyed  by  boiling.  It  disappears 
from  yeast-juice  during  fermentation  and  when  the  juice  is  allowed  to 
undergo  autolysis.  The  evolution  of  carbon  dioxide  from  a  mixture  to 
which  a  small  amount  of  co-enzyme  has  been  added  soon  ceases,  but 
can  be  renewed  by  the  addition  of  more  co-enzyme.  A£  to  the  nature 
of  this  body  our  knowledge  is  as  yet  incomplete.  In  view  of  the  fact 
that  soluble  inorganic  phosphates  are  able  to  greatly  increase  the 
activity  of  an  ordinary  yeast-juice,  it  was  thought  that  these  substances 
might  be  the  co-enzyme.  Experiments  showed,  however,  that  the  in- 
active residue  could  not  be  brought  back  to  activity  by  this  means, 

65  5 


66  THE  NATURE  OF  ENZYME-ACTION 

although  subsequent  addition  of  boiled  juice  was  able  to  do  so.  More- 
over, the  boiled  autolysed  juice  does  not  set  up  fermentation  in  a 
mixture  of  the  inactive  residue  with  glucose,  although  it  contains  a 
large  amount  of  soluble  phosphate.  Subsequent  work  (Part  III.) 
showed  that  both  the  bodies  mentioned  are  necessary  for  the  activity 
of  zymase.  The  system  is,  therefore,  a  very  complex  one,  two  co- 
enzymes,  in  fact,  being  required.  Phosphates  are  necessary,  but  in- 
effective apart  from  the  presence,  in  addition,  of  another  co-enzyme, 
whose  nature  is,  as  yet,  unknown.  "  The  cycle  of  changes  which  the 
phosphate  undergoes  appears  to  be  the  following : — 

(1)  2C6H1206  +  2Na2HP04  =  2CO2  +  2C2H6O  +  C6H10O4(PO4Na.2).2 
+  2H20 

(2)  C6H1004(P04Na2)2  +  2H20  =  C6H12O6  +  2Na2HPO4." 

These  changes,  of  course,  take  place  under  the  influence  of  the 
enzyme-system. 

The  chemical  nature  of  the  lipase  co-enzyme  is  better  known  than 
that  of  zymase.  Loevenhart  (112)  showed  that  bile-salts  possessed  all 
the  properties  of  the  co-enzyme,  while  v.  Furth  and  Schutz  (68)  have 
shown  that  sodium  cholate  is  as  active  as  sodium  glycocholate. 

Magnus  (183)  also  showed  that  synthetic  bile-salts  have  the  same 
action  as  the  natural  bodies.  The  importance  of  this  fact  is  that  it 
shows  that,  unlike  the  action  of  phosphate  on  yeast-juice,  no  additional 
co-enzyme  is  required,  such  as  might  possibly  be  contained  in  prepara- 
tions of  the  natural  bile-salts. 

One  more  instance  may  be  referred  to.  It  has  been  shown  by 
Bierry,  Giaja  and  V.  Henri  (28)  that  if  pancreatic  juice  be  dialysed,  it 
loses  its  power  of  acting  upon  starch  or  maltose.  The  addition  of  cer- 
tain electrolytes  restores  this  activity.  By  testing  various  salts  it  was 
shown  that  the  electro-negative  ion  is  the  only  potent  one,  and  among 
these  ions  the  chlorine  or  bromine  ion  is  the  essential  one.  Thus, 
sodium  and  potassium  chlorides  are  active,  while  the  sulphates  are  in- 
active. 

There  are  also,  in  contradistinction  to  the  bodies  treated  of  above, 
certain  other  bodies  'which  hinder  the  action  of  enzymes  in  a  specific 
manner.  These  "anti-enzymes"  are  similar  to  those  anti-toxins  pro- 
duced by  the  injection  of  toxins  into  the  living  organism,  so  that  we 
may  regard  enzymes  as  belonging  to  that  class  of  bodies  which  act  in 
minute  amounts  and  which  Ehrlich  (55)  considers  to  be  similar  to 
food-stuffs  and  taken  up  by  the  protoplasm  of  living  cells  in  some  inti- 
mate connection.  The  other  class  of  bodies  act  by  the  physical  or 
chemical  characters  of  their  molecules  and  do  not  give  rise  to  anti- 
bodies when  injected.  Such  bodies  are  of  small  molecular  weight,  and 
in  this  class  are  included  drugs  in  general  and  the  chemical  messengers 
or  hormones  (20,  p.  668,  and  149). 

Several  anti-enzymes  are  normally  present  in  the  blood,  such  as 
anti-trypsin  and  anti-rennet,  others  can  be  produced  by  the  injection  of 
enzymes  subcutaneously ;  by  this  latter  means  anti-bodies  to  the  follow- 
ing enzymes  have  been  obtained — lipase,  emulsin,  amylase,  pepsin, 
papain  and  urease. 

A  very  interesting  anti-trypsin  was  found  by  Weinland  (165)  in 
intestinal  worms,  which  seems  to  have  the  function  of  protecting  them 
from  the  action  of  the  pancreatic  juice.  The  properties  of  this  body 


CO-ENZYMES  AND  ANTI-ENZYMES 


67 


were  investigated  by  J.  M.  Hamill  (71).  It  is  not  destroyed  by  boiling 
in  neutral  or  acid  solutions ;  but,  if  made  even  faintly  alkaline,  its  anti- 
action  is  immediately  destroyed  on  boiling.  It  is  soluble  in  alcohol  of 
strengths  below  85  per  cent,  by  stronger  alcohol  it  is  precipitated  un- 
injured. It  dialyses  readily  through  colloid  membranes. 

In  the  course  of  the  reaction  anti-trypsin  slowly  disappears,  as  was 
shown  by  Dastre  (46),  for  the  maceration  of  intestinal  worms  and  which 
I  have  been  able  to  confirm.  If  a  mixture  of  trypsin  and  caseinogen, 
to  which  sufficient  worm-extract  has  been  added  to  inhibit  the  reaction, 
be  allowed  to  remain  in  the  thermostat  for  a  few  days,  it  will  be  found 
that  the  enzyme  gradually  becomes  active  again. 

If  raw  serum  or  egg-albumin  containing  anti-trypsin  be  acted  upon 
by  trypsin,  it  will  be  found  that  for  some  hours  no  effect  will  be  pro- 
duced, but  that  gradually  the  trypsin  begins  to  regain  its  activity  and 


10 


20  30          40  50 

TIME  IN  HOURS. 

FIG.  7. 


60 


in  such  a  way  that  the  curve  of  rate  of  change  is  convex  to  the  axis 
of  abscissae,  showing  that  the  recovery  of  the  enzyme  is  a  gradual  one 
and  the  effect  is  not  to  be  ascribed  to  difficulty  of  attack  on  the  part 
of  the  protein  itself.  Fig.  7  shows  the  phenomenon  in  question.  The 
upper  curve  is  that  of  egg-white,  diluted  with  nine  times  its  volume  of 
water,  and,  after  heating  to  100°,  in  order  to  destroy  the  anti-body,  acted 
on  by  trypsin.  The  lower  curve  is  that  of  a  similar  mixture,  but  in 
which  the  egg-white  had  not  been  heated.  The  anti-body  was  at  first 
intact  in  this  latter  solution,  but  it  gradually  disappeared,  so  that  finally 
the  same  amount  of  change  had  taken  place  in  both  the  digests. 

The  manner  in  which  enzymes  are  rendered  inactive  by  combina- 
tion with  their  anti-bodies  is  regarded  by  Hedin  (179)  as  being  similar 
to  that  shown  by  him  to  obtain  in  the  case  of  the  taking  up  of  trypsin 
by  charcoal,  that  is,  by  an  adsorption-process.  The  two  characteristic 

5* 


68  THE  NATURE  OF  ENZYME-ACTION 

phenomena  common  to  the  two  cases  are  (i)  it  is  impossible  to  take  up 
all  the  trypsin  out  of  a  solution  even  by  excess  of  the  anti-body, 
whether  it  be  charcoal  or  that  present  in  serum,  and  (2)  relatively  more 
trypsin  is  neutralised  by  small  amounts  of  the  anti-body  than  by  larger. 
The  process,  as  far  as  charcoal  is  concerned,  consists  of  two  stages,  the 
enzyme  is  first  taken  up  in  such  a  form  that  it  is  readily,  in  great  part, 
given  up  again  to  caseinogen ;  the  second  stage  appears  to  be  a  kind  of 
fixation,  so  that  it  is  not  so  given  up.  There  is  no  sharp  line  of  de- 
marcation between  the  two  processes,  and  it  seems  possible  that  the 
second  stage  may  be  similar  to  the  diffusion  of  iodine  into  the  interior 
of  the  carbon  in  the  researches  of  Davis  (181).  The  specific  nature  of 
certain  anti-bodies  is  no  argument  against  the  view  of  Hedin,  as  has 
been  shown  in  the  previous  chapter. 

Weinland  (166)  has  stated  that  anti-pepsin  exists  in  the  gastric 
mucous  membrane  and  anti-trypsin  in  that  of  the  intestine ;  these  bodies 
are  supposed  to  confer  upon  these  tissues  their  immunity  from  attack 
by  the  digestive  juices.  According  to  some  recent  work  of  Hamill  as 
well  as  of  myself,  the  existence  of  anti-trypsin  in  the  intestinal  mucous 
membrane  is  very  doubtful,  although  Hamill  confirms  the  presence  of 
anti-pepsin  in  the  gastric  mucous  membrane. 

According  to  Klug  (176)  the  actual  body  which  has  the  power  of 
protecting  the  cells  of  the  mucous  membrane  is  the  mucin  which  is 
always  present  in  considerable  quantity.  This  it  does  by  forming 
adsorption-compounds  with  the  enzymes. 

If  this  be  so,  it  may  reasonably  be  extended  to  the  other  colloids 
present  in  the  foods  taken  into  the  alimentary  canal.  It  is  to  be  pre- 
sumed that,  by  mass-action,  the  greater  part  of  the  enzymes  is  in  com- 
bination with  these  substances.  It  is  certain  that,  if  a  copious  secretion 
of  pancreatic  juice  is  poured  into  the  empty  intestine  in  consequence  of 
the  injection  of  secretin,  great  damage  is  done  to  the  mucous  membrane, 
resulting  in  desquamation  of  the  cells  and  haemorrhage.  No  doubt, 
under  normal  conditions,  the  acid  gastric  contents,  which  serve  to  excite 
the  secretion  of  the  pancreatic  juice,  when  they  arrive  in  the  duodenum, 
also  effect  a  partial  neutralisation  of  its  strong  alkalinity,  a  factor  which 
is  absent  when  the  pancreatic  secretion  is  excited  by  the  injection  of 
ready-made  secretin.  At  the  same  time,  it  is  obvious  that  the  re- 
action must  remain  sufficiently  alkaline  in  order  that  the  trypsin  may 
exert  its  action.  It  seems  probable,  then,  that  the  injurious  action  of 
pancreatic  juice  on  the  empty  intestine  may  be,  in  part,  due  to  the 
absence  of  food-stuffs,  which  would  take  up  the  enzyme. 


CHAPTER  IX. 

ZYMOGENS. 

THE  relation  between  enzyme  and  co-enzyme  is  a  reversible  one. 
This  can  be  seen  by  considering  the  case  of  the  liver  lipase.  The 
extracts  were  at  first  active,  became  inactive  on  dialysis,  but  regained 
activity  on  the  addition  of  bile-salts. 

The  relation  of  inactive  zymogen  to  active  enzyme  is  an  irrever- 
sible one.  Since  all  enzymes  are  produced  by  the  agency  of  living 
protoplasm,  it  is  evident  that  at  some  stages  in  their  formation  they 
must  be  devoid  of  the  catalytic  properties  of  the  fully  formed  enzyme. 
This  stage  is  called  a  "  zymogen  "  when  it  can  be  obtained  free  from 
the  cells  in  which  it  was  formed  and  can  be  converted  by  purely 
chemical  means  into  the  active  enzyme.  When  this  change  has  taken 
place  the  new  body  cannot,  as  far  as  we  know,  be  reconverted  into 
the  zymogen. 

The  trypsin  of  the  pancreatic  juice  is  actually  secreted  in  the  form 
of  a  zymogen  and  is  poured  into  the  duodenum  in  this  state,  as  has 
been  shown  by  the  present  author  in  conjunction  with  Starling  (17,  p. 
347).  In  the  duodenum  it  meets  with  the  enzyme,  enterokinase,  and 
is  converted  by  this  into  active  trypsin.  Except  for  the  fact  of  its  being 
devoid  of  proteoclastic  power,  trypsinogen,  as  we  may  call  the  zymogen 
of  trypsin,  has  properties  very  like  those  of  the  enzyme. 

Recent  experiments  of  Delezenne  (47)  show  that  inactive  pan- 
creatic juice  can  be  activated  by  calcium  salts  as  well  as  by  entero- 
kinase. The  amount  of  calcium  present  in  the  juice  as  secreted  is 
sufficient  to  bring  about  very  slow  activation,  but  the  process  can  be 
considerably  accelerated  by  adding  more  calcium.  These  results  have 
been  confirmed  by  Miss  B.  Ayrton,  in  the  Physiological  Laboratory 
of  University  College,  London,  but  not  yet  published. 

The  first  preparations  containing  a  zymogen  in  solution  were  those 
of  Langley  and  Edkins  (108).  These  solutions  contained  a  substance, 
pepsinogen,  which,  on  treatment  with  hydrochloric  acid,  was  converted 
into  active  pepsin.  The  proof  of  the  existence  of  a  zymogen  in  extracts 
containing  pepsin  itself  was  rendered  possible  by  the  discovery  of 
Langley  (107,  p.  253)  that  pepsin  is  much  more  rapidly  destroyed  by 
alkali  than  is  pepsinogen.  The  latter  is  very  rapidly  converted  into 
pepsin  by  acids ;  at  20°  all  or  nearly  all  the  pepsinogen  present  in  an 
aqueous  extract  of  a  cat's  gastric  mucous  membrane  may  be  converted 
into  pepsin  in  sixty  seconds  by  O'l  per  cent,  hydrochldric  acid. 

Pepsinogen  has  been  prepared  by  Glaessner  (69)  in  what  appears 
to  have  been  a  nearly  pure  solution  by  a  method  of  which  the  follow- 
ing is  an  outline.  The  mucous  membrane  of  pigs'  stomachs  was  allowed 
to  autolyse  in  alkaline  solution  at  40°  for  some  weeks.  Mucin  was 

69 


70  THE  NATURE  OF  ENZYME-ACTION 

removed  by  precipitation  with  acetic  acid  and  proteins  with  uranyl- 
acetate ;  this  last  precipitate  carried  down  with  it,  in  a  state  'of  ad- 
sorption, the  pepsinogen.  By  extracting  the  mass  with  a  large  amount 
of  dilute  sodium  carbonate  the  greater  part  of  the  zymogen  passed 
into  solution  again.  The  process  of  uranyl-acetate  precipitation  and 
extraction  of  precipitate  with  dilute  sodium  carbonate  was  repeated 
and  the  final  extracts  concentrated  at  40°. 

The  body  so  obtained  gave  only  two  of  the  protein  reactions  and 
these  very  faintly,  namely,  a  fine  precipitate  with  mercuric  salts  and 
with  phosphotungstic  acid.  It  must  therefore  be  of  non-protein  nature, 
and,  if  so,  the  pepsin  formed  from  it  by  the  action  of  acid  can  scarcely 
be  a  protein. 

The  physical  properties  of  the  substance  are  interesting.  It  was 
found  to  be  slightly  laevo-rotatory.  We  have  already  seen  reason  to 
suppose  that  enzymes  are  optically  active.  It  is  adsorbed  by  kieselguhr, 
alumina  and  fibrin,  but  not  by  starch  or  sand.  These  facts  serve  to  con- 
firm the  view  of  the  specific  nature  of  adsorption,  since  it  can  scarcely 
be  held  that  pepsinogen  has  a  greater  chemical  affinity  for  kieselguhr 
than  it  has  for  sand,  although  it  would  be  expected  to  have  more 
"  affinity  "  for  fibrin  than  for  starch. 

It  is  of  interest  to  notice  the  close  similarity  between  the  enzyme 
and  its  zymogen,  at  all  events  in  the  present  case.  As  Glaessner  re- 
marks, the  conversion  by  very  dilute  mineral  acids  with  such  smooth- 
ness, rapidity  and  completeness  is  only  explicable  on  the  assumption 
of  a  very  simple  chemical  process. 


CHAPTER  X. 

OXIDATION-PROCESSES  AND  CERTAIN  COMPLEX  SYSTEMS. 

INSTANCES  have  been  given  in  the  preceding  pages  where  a  system 
consisting  only  of  enzyme  and  substrate  undergoes  no  perceptible  change. 
The  addition  of  a  third  body  is  requisite  in  order  that  a  reaction  may 
take  place.  Such  cases  were  those  of  lipase  and  zymase  with  their  re- 
spective co-enzymes,  as  also  pepsin  and  trypsin  with  acid  and  alkali 
respectively. 

A  different  case  is  that  of  the  various  enzymes  concerned  in  the 
mechanism  of  oxidation.  It  will  probably  have  been  noticed  that  the 
enzyme-processes  dealt  with  in  any  detail  up  to  the  present  were  all 
hydrolytic  actions.  The  intimate  mechanism  of  the  oxidation  enzymes 
is  still  comparatively  little  understood,  and  until  the  work  of  Bach  and 
Chodat(n)  it  was  involved  in  much  confusion.  Although  the  point 
of  view  of  these  investigators  may  not  as  yet  explain  all  the  phenomena 
met  with  in  this  difficult  subject,  it  is  by  far  the  most  satisfactory  hypo- 
thesis hitherto  propounded. 

In  the  consideration  of  the  subject  there  are  three  distinct  sets  of 
bodies  to  be  taken  into  account.     These  are  : — 
^>         I.  Organic  peroxides,  including  hydrogen  peroxide. 

2.  Peroxydases. 

3.  Catalases. 

^The  two  last  only  are  of  enzyme  nature.  They  both  decompose 
hydrogen  peroxide  with  formation  of  water  and  oxygen,  but  while  the 
peroxydase  separates  oxygen  in  the  active  state,  probably  as  atomic 
oxygen,  the  catalase  separates  it  as  molecular  or  relatively  inactive 
oxygen.  The  catalase  must  therefore  attack  two  molecules  of  the 
peroxide  simultaneously,  since  only  one  atom  of  oxygen  is  afforded  by 
each  molecule  of  peroxide.  A  second  point  of  difference  is  that  while 
catalase  acts  only  on  hydrogen  peroxide,  the  peroxydases  act  on  various 
organic  peroxides ;  at  any  rate  their  general  action  can  only  be  ex- 
plained on  this  hypothesis,  and  Bach  and  Chodat  have  shown  that  ethyl- 
hydrogen  peroxide  is  split  by  them. 

^~  Now  molecular  oxygen  is  incapable  of  acting  upon  the  greater 
number  of  bodies,  such  as  glucose,  lactic  acid  or  uric  acid,  which  are  oxi- 
dised in  the  organism,  so  that,  although  catalase  is  very  widespread,  its 
function  is  somewhat  problematic,  except  in  the  case  of  the  green  plant, 
asiwe  shall  see  later.  It  is  possible  that  hydrogen  peroxide  may  be 
produced  as  a  bye-product  of  oxidations  in  the  animal  tissues ;  if  so,  it 
is  necessary  that  it  should  be  forthwith  destroyed,  owing  to  its  toxic 
action  on  living  protoplasm. 

„  A  peroxydase  alone,  without  the  presence  of  a  peroxide,  is  obviously 

of  no  use  as  an  oxidising  agent,  so  that  the  system  of  peroxide  and 

71 


72  THE  NATURE  OF  ENZYME-ACTION 

peroxydase  is  the  active  combination.  This  system  is,  in  fact,  what  is 
sometimes  called  an  "  oxydase  ".  In  order  that  the  process  of  oxidation 
may  be  continuous,  the  peroxide  component  of  the  system  must  be 
capable  of  re-formation  by  taking  up  oxygen  again.  Accordingly  we 
find  that  the  oxidation  of  guaiacum  by  potatoes,  for  example,  does  not 
take  place  in  the  absence  of  oxygen. 

An  instructive  experiment  consists  in  the  use  of  peroxydase,  such  as 
that  to  be  obtained  from  the  root  of  the  horse-radish,  instead  of  an  in- 
organic catalyst  in  Fenton's  reaction.  If  a  mixture  of  lactic  acid  and 
hydrogen  peroxide  be  taken,  it  will  be  found  that  oxidation  proceeds 
with  extreme  slowness.  A  trace  of  ferrous  sulphate  will  enormously 
accelerate  the  reaction.  If  the  peroxydase  of  horse-radish  be  added 
instead  of  the  iron  salt,  it  is  found  that  the  reaction  is  accelerated  in  the 
same  way.  In  other  words,  peroxydase  is  capable  of  taking  the  part 
of  the  iron  salt  in  this  reaction.  This  fact,  in  conjunction  with  that  of 
the  powerful  effect  of  the  addition  of  manganese  or  iron  to  various 
c<  oxydases,"  and  the  apparently  universal  occurrence  of  one  or  other  of 
these  bodies  in  the  ash  of  these  enzymes,  suggests  that  the  latter  may 
be  a  means  of  rendering  iron,  manganese  or  a  similar  body  in  an  oxidis- 
able  system  into  an  extremely  active  state.  This  suggestion  remains 
as  yet,  however,  in  the  region  of  pure  hypothesis. 

The  chief  difficulty  in  the  universal  application  of  the  Bach-Chodat 
theory  lies  in  the  very  specific  nature  of  many  oxydases.  Laccase  was 
shown  by  Bertrand  (25)  to  act  upon  hydroquinone  or  pyrogallol  with 
great  facility,  but  not  at  all  on  resorcinol  or  phloroglucinol.  Moreover, 
it  does  not  act  upon  tyrosine,  which  is  readily  oxidised  by  another 
enzyme  "  tyrosinase ".  A  satisfactory  explanation  of  these  facts  is 
wanting.  It  would  be  expected  that  active  oxygen  liberated  by  the 
agency  of  a  peroxydase  would  be  able  to  effect  oxidations  indiscrimin- 
ately, and  the  experiment  on  lactic  acid  and  hydrogen  peroxide  cited 
above  seems  to  indicate  that  the  specificity  lies  in  the  peroxide  com- 
ponent of  the  oxydase  system.  If  this  be  so,  it  is  possible  that  there 
is  some  intimate  relationship  necessary  between  the  structure  of  the  per- 
oxide and  the  substrate  in  order  that  close  connection  may  be  possible 
so  that  the  active  oxygen  may  enter  into  immediate  union  with  the  latter. 

There  is  an  interesting  class  of  chemical  reactions  called  by  Ostwald 
(128)  "  coupled  reactions,"  which  it  seems  probable  will  have  to  be 
taken  into  account  in  oxidation,  and  perhaps  also  in  other  processes  in 
the  organism.  An  instance  of  such  a  reaction  is  the  following :  When 
a  solution  of  sodium  arsenite  is  shaken  with  air  no  oxidation  takes 
place ;  sodium  sulphite  similarly  treated  is  rapidly  oxidised ;  if  a 
mixture  of  the  two  be  shaken  with  air  both  salts  are  oxidised.  In  some 
way  or  other  oxygen  is  activated  in  the  process.  It  is  convenient  to 
give  names  to  reactions  in  which  a  slow  reaction  is  accelerated  by  the 
existence  of  a  simultaneous  rapid  reaction  between  one  of  the  reacting 
bodies  of  the  former  reaction  and  a  third  body.  The  rapid  reaction  is 
called  the  "  primary  reaction,"  and  that  which  is  induced  by  the  pre- 
sence of  the  former  is  called  the  "  secondary  reaction  ".  The  body 
common  to  both  is  called  the  "  actor " ;  the  second  substance  in  the 
primary  reaction  is  the  "  inductor "  ;  and  the  corresponding  body  in 
the  secondary  reaction  is  the  "  acceptor ".  In  the  example  given, 
oxygen  is  the  actor,  sodium  sulphite  the  inductor,  and  sodium  arsenite 


OXIDATION-PROCESSES  AND  COMPLEX  SYSTEMS        73 

the  acceptor.  Certain  catalytic  reactions  already  referred  to  may  be 
described  from  this  point  of  view ;  the  catalysis  by  ferrous  sulphate  of 
the  oxidation  of  hydriodic  acid  by  hydrogen  peroxide  is  one  of  these. 
In  this  process,  the  reaction  FeSO4  +  H2O2  is  the  primary  reaction, 
H2O2  +  HI  is  the  secondary  reaction,  H2O2  is  the  actor,  FeSO^  the  in- 
ductor and  HI  is  the  acceptor.  For  further  information  on  this  ques- 
tion, as  yet  imperfectly  worked  out,  the  reader  is  referred  to  Mellor's 
Chemical  Statics  and  Dynamics. 

In  connection  with  the  properties  of  catalase,  the  researches  of 
Usher  and  Priestly  (158)  on  the  chlorophyll-function  are  of  interest. 
According  to  these  observers,  this  system  consists  of  three  partners — the 
protoplasm  of  the  chloroplast,  the  chlorophyll  itself  and  a  catalase.  By 
means  of  the  pigment,  acting  as  both  optical  and  chemical  sensitiser, 
light-energy  is  employed  to  cause  reaction  between  carbon  dioxide  and 
water  in  such  a  manner  that  formaldehyde  and  hydrogen  peroxide  are 
formed.  Both  of  these  bodies  are  toxic,  and,  if  allowed  to  accumulate, 
the  reaction  soon  comes  to  an  end.  The  formaldehyde  is,  however, 
rapidly  polymerised  by  the  protoplasm  of  the  chloroplast  and  the  hydrogen 
peroxide  is  split  into  oxygen  and  water  by  the  catalase.  We  see  thus 
why  the  reaction  as  a  whole  does  not  occur  in  non-living  preparations 
or  extracts  of  green  leaves.  Formaldehyde  is  indeed  produced  in  the 
presence  of  chloroform  on  exposure  to  light  of  appropriate  arrangements 
of  the  pigment,  but  since  no  polymerisation  occurs,  the  chlorophyll  is 
destroyed  by  it  and  the  reaction  comes  to  an  end.  Production  of  hydro- 
gen peroxide  and  formaldehyde  also  takes  place  in  leaves  killed  by  boil- 
ing, and  in  this  case,  since  the  enzyme,  catalase,  is  destroyed,  as  well  as 
the  protoplasm,  the  hydrogen  peroxide  also  contributes  to  the  destruc- 
tion of  the  chlorophyll.  It  is  well  to  mention  that  some  doubt  has  re- 
cently been  thrown  on  these  results  by  Ewart  (61). 

The  factors  concerned  in  the  coagulation  of  the  blood  form  another 
extraordinarily  complex  system.  According  to  Morawitz  (122,  123), 
there  exists  in  circulating  blood  a  body,  thrombogen,  which  can  be  con- 
verted by  a  thrombokinase,  present  in  all  tissues,1  including  the  formed 
elements  of  the  blood,  into  a  precursor  of  the  enzyme  which  acts  upon 
fibrinogen  to  form  fibrin.  This  precursor,  or  prothrombase,  is  changed 
into  the  active  thrombase  by  calcium  ions.  According  to  Nolf  (i  24),  the 
process  consists  essentially  in  the  interaction  of  three  colloidal  proteins  in 
the  presence  of  calcium  ions.  These  proteins  are  thrombogen,  throm- 
bozyme  and  fibrinogen.  Both  fibrin  and  thrombase  consist  of  all  three 
of  these  colloids,  but  in  different  proportion  ;  fibrin  contains  fibrinogen 
in  excess,  a  circumstance  which  accounts  for  its  insolubility.  Throm- 
bozyme  is  a  proteolytic  enzyme,  the  process  of  coagulation  being  the 
first  stage  of  its  action,  while  the  well-known  fibrinolysis  is  the  second 
stage.  This  brief  statement  gives  but  a  mere  abstract  of  this  important 
work  which  is,  in  the  main,  a  confirmation  and  extension  of  that  of  Wool- 
dridge.  For  further  details  the  original  papers  must  be  consulted. 

Finally,  under  the  heading  of  this  chapter,  may  be  mentioned  those 
fairly  numerous  cases  where  more  than  one  enzyme  is  requisite  to  effect 
a  particular  change.  Here  the  enzymes  act  one  after  the  other  and  the 
products  of  the  one  become  the  substrate  of  the  next.  According  to 

1  With  respect  to  the  properties  of  tissue-extracts,  the  paper  of  Pekelharing  (188)  should 
be  consulted. 


74  THE  NATURE  OF  ENZYME-ACTION 

Buchner  and  Meisenheimer  (42),  the  system 'responsible  for  the  alcoholic 
fermentation  of  sugar  consists  of  two  distinct  enzymes ;  the  first  con- 
verts sugar  into  lactic  acid,  and  is  the  zymase  proper ;  it  has  also  been 
prepared  by  these  investigators  from  the  lactic  acid  bacillus.  The  sec- 
ond enzyme,  lactacidase,  in  turn  acts  upon  the  lactic  acid,  converting  it 
into  alcohol  and  carbon  dioxide. 

Another  interesting  case  is  that  of  the  enzymatic  hydrolysis  of  gen- 
tianose,  as  investigated  by  Bourquelot  (31)  and  Herissey  (32).  This 
sugar  is  a  tri-hexose,  consisting  of  two  molecules  of  glucose  and  one  of 
fructose.  If  it  be  subjected  to  the  action  of  invertase  the  fructose  is 
split  ofif,  leaving  the  two  molecules  of  glucose  still  united  as  a  bi-hexose. 
Emulsin,  or  rather  the  mixture  of  enzymes  obtained  from  the  bitter- 
almond,  is  able  now  to  split  this  bi-hexose  into  its  components.  The  im- 
portance of  this  case  is  that  if  a  mixture  of  emulsin  and  invertase  had  been 
added  at  once  the  gentianose  would  have  been  completely  hydrolysed  ; 
the  juice  of  Aspergillus  appears  to  contain  such  a  mixture,  which  might 
have  been  mistaken  for  a  single  enzyme  but  for  the  previous  experi- 
ments. It  has  recently  been  shown  by  Armstrong  and  Horton  (178) 
that  the  "emulsin"  of  the  almond  contains  three  distinct  enzymes. 
These  are,  a  lactase,  a  /3-glucase,  which  causes  the  hydrolysis  of  /3-glu- 
cosides,  such  as  salicin,  while  the  third  is  an  enzyme  which  separates 
glucose  from  amygdalin,  leaving  Fischer's  amygdonitrile-glucoside. 

Raffinose,  a  tri-hexose  consisting  of  glucose,  fructose  and  galactose, 
as  shown  by  Fischer  and  Lindner  (66),  requires  the  action  of  a  series  of 
enzymes  to  effect  its  complete  hydrolysis.  According  to  Armstrong 
and  Glover  (177),  when  hydrolysed  by  acids  or  invertase,  it  is  resolved 
into  fructose  and  melibiose,  while  the  lactase  of  "  emulsin  "  converts  it 
into  cane-sugar  and  galactose,  as  first  shown  by  Neuberg  (189). 

For  the  conversion  of  starch  into  glucose,  as  we  have  already  seen, 
three  enzymes  are  necessary.  Amylase  transforms  it  into  dextrins, 
which  are  then  converted  by  the  dextrinase  of  Duclaux  into  maltose, 
which  is  finally  changed  to  glucose  by  maltase. 

The  same  kind  of  phenomena  are  observed  in  the  series  of  pro- 
teoclastic  enzymes  met  with  by  the  food  as  it  traverses  the  alimentary 
canal.  Erepsin  acts  upon  the  products  of  the  action  of  trypsin,  while 
this  latter  enzyme  itself  acts  upon  the  products  of  peptic  digestion. 


GENERAL  CONCLUSIONS. 

THE  living  organism  is  enabled  by  the  use  of  enzymes  to  bring  about, 
under  ordinary  conditions  of  temperature  and  moderate  concentrations 
of  acid  or  alkali,  many  chemical  reactions  which  would  otherwise  re- 
quire a  high  temperature  or  powerful  reagents. 

A  careful  study  of  these  enzymes  shows  that  they  obey  the  usual 
laws  of  catalytic  phenomena.  Certain  deviations  from  the  behaviour 
of  most  inorganic  catalysts  are  found  to  depend  upon  the  colloidal 
nature  of  enzymes,  so  that  the  reactions  take  place  in  a  heterogeneous 
medium  and  the  various  phenomena  depending  upon  surface  action 
come  markedly  into  play. 

As  they  are  sensitive  to  heat  and  more  or  less  rapidly  destroyed  by 
it,  they  show  the  phenomenon  of  a  so-called  optimun  temperature. 
This  destruction  by  heat  is,  in  all  probability,  due  to  their  organic 
colloidal  nature. 

So  far  as  we  know,  the  reactions  catalysed  by  enzymes  are  rever- 
sible in  nature,  but  since,  as  investigated  in  vitro,  they  take  place  in 
presence  of  excess  of  water,  the  equilibrium  position  is  usually  very 
near  the  stage  of  complete  hydrolysis.  Owing  to  this  reversible  char- 
acter of  the  reactions,  it  follows  that  enzymes  have  synthetic  action. 

Reasons  are  given  for  the  belief  that  the  "  compound  "  of  enzyme  and 
substrate,  generally  regarded  as  the  preliminary  to  action,  is  of  the 
nature  of  a  colloidal  adsorption-compound. 

The  existence  of  a  relation  of  this  kind  explains  the  exponential  form 
of  the  law  correlating  the  concentration  of  enzyme  with  its  activity. 

Autocatalysis,  positive  and  negative,  plays  a  considerable  part  in  the 
changes  of  activity  of  an  enzyme  during  the  course  of  its  action.  These 
changes  of  "  activity  "  are  the  main  factors  in  the  deviations  of  the  form 
of  the  equation  for  the  velocity  of  reaction  from  the  simple  unimole- 
cular  formula,  when  the  reaction,  as  is  usually  the  case,  takes  place  in 
presence  of  excess  of  water. 


75 


SUPPLEMENTARY  NOTES. 

NOTE  A. 

IN  illustration  of  the  great  activity  of  the  organic  agents  or  enzymes  which  effect  these 
changes,  as  compared  with  inorganic  hydrolytic  agents,  the  following  experiment  of  Frank- 
land  Armstrong  (5,  iv.,  p.  533)  may  be  quoted  :  A  preparation  of  lactase  was  found  to 
hydrolyse  about  one-fourth  of  the  milk-sugar  contained  in  a  5  per  cent,  solution  in  one  hour 
at  35°,  whereas  a  twice  normal  hydrochloric  acid  required,  at  the  same  temperature,  about 
five  weeks  to  effect  the  same  amount  of  hydrolysis. 

NOTE  B. 

It  should  be  mentioned  that  this  formation  of  amino-acids  (Lawrow  [109,  p.  516])  is 
regarded  by  some  as  being  due  to  the  presence  of  autolytic  enzyme  in  ordinary  pepsin 
preparations.  This  does  not  seem  very  probable,  however,  in  view  of  the  difficulty  of 
obtaining  solutions  of  autolytic  enzymes,  owing  to  their  intracellular  nature.  It  is  desir- 
able that  the  experiments  be  repeated  with  natural  gastric  juice.  It  will  be  remembered 
that  Fischer  and  Abderhalden  (65,  p.  55)  found  that  the  pancreatin  "  Rhenania"  which  ap- 
pears to  be  dried  pancreas,  was  able  to  hydrolyse  leucyl-alanine  to  some  degree,  whereas 
fresh  pancreatic  juice  was  unable  to  do  so.  Dried  pancreas  would  probably  contain  a 
small  amount  of  autolytic  enzyme. 

NOTE  C. 

The  following  remarks  by  van't  Hoff  (90,  p.  12)  will  be  read  with  interest:  "The, 
theory  of  chemical  equilibrium  may  also  find  its  application  here  (i.e.,  in  organic  chemistry) 
and  indeed  has  already  done  so ;  on  account  of  the  great  variety  of  compounds  and  the 
inertia  of  reaction,  however,  appropriate  choice  of  material  is  not  easy.  It  is,  perhaps,  on 
this  account,  of  some  value  to  direct  attention  to  the  extremely  noteworthy  ferment-  or 
enzyme-actions,  which  have  shown  themselves  admirable  for  this  purpose,  as  recent  in- 
vestigations demonstrate.  On  the  one  hand,  Fischer  (63)  found  that,  under  the  action  of 
ferments,  organic  changes  are  directed  into  determinate  paths,  a  fact  which  completely 
excludes  complication  by  variety  of  forms.  On  the  other  hand,  the  recent  researches  of 
Tammann  (152-154),  Duclaux  (51)  and  especially  of  Hill  (86)  cannot  be  explained  without 
the  introduction  of  considerations  of  equilibrium.  It  was  pointed  out  by  Tammann  that, 
under  the  action  of  emulsin,  amygdalin  is  only  partially  split  and  that  this  hydrolysis  pro- 
ceeds further  if  the  products  are  removed.  Perhaps  if  he  had  added  a  further  amount  of 
products  of  hydrolysis,  he  might  have  succeeded  in  synthesising  amygdalin.  Duclaux  put 
forward  transformation  formulae,  which  again  suggest  the  attainment  of  an  equilibrium, 
and  Hill  seems  to  have  effected  the  synthesis  of  maltose  from  glucose  by  means  of  a  yeast 
enzyme.  Unless  a  ferment  undergoes  alteration  of  some  kind  during  its  period  of  activity, 
it  follows,  on  theoretical  grounds,  that  a  condition  of  equilibrium  and  not  one  of  total 
change  must  be  brought  about,  and  that  therefore  the  opposite  reaction  must  be  induced. 
We  are  indeed  justified  in  asking  the  question,  whether  (by  application  of  the  theory  of 
equilibrium),  under  the  influence  of  zymase  and  by  exceeding  a  certain  limiting  opposing 
pressure  of  carbon  dioxide,  glucose  might  not  be  formed  from  alcohol  and  carbon  dioxide, 
and  moreover  whether  trypsin  may  not  be  able,  under  conditions  prescribed  by  the  theory 
of  equilibrium,  to  form  protein  from  the  products  of  the  hydrolysis  which  it  brings  about  under 
other  conditions." 

76 


SUPPLEMENTARY  NOTES  77 

NOTE  D. 

Enzymes  do  not  differ  from  chemical  catalysts  of  known  composition,  in  their  behaviour 
towards  optical  isomers,  as  has  been  shown  recently  by  Bredig  and  Fajans  (34).  The  d- 
and  1-camphor-carboxylic  acids  in  solution  in  acetophenone  slowly  decompose  with  evolu- 
tion of  CO2.  This  reaction  is  catalysed  by  bases.  When  an  optically  inactive  base  is 
used,  both  acids  are  decomposed  at  equal  rates,  but  if  an  optically  active  base,  such  as 
nicotine,  be  used,  it  is  found  that  the  d-acid  is  acted  upon  at  a  rate  which  is  17  per  cent, 
faster  than  that  at  which  the  other  acid  is  decomposed.  As  ordinary  nicotine  is  laevo- 
rotatory,  it  would  be  of  much  interest  to  know  whether  the  dextro-rotatory  nicotine  would 
decompose  the  1-acid  faster  than  the  d-acid. 


LIST  OF  LITERATURE  REFERRED  TO. 

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6 


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84.  Henry,  P.,  "  Ueber  die  wechselseitige  Umwandlung  der  Laktone  und  der  Oxy- 

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INDEX. 


ACCELERATION  of  action,  causes  of,  46,  49. 
Accelerators  contrasted  with  co-enzymes,  65. 
Acetone  as  precipitant  and  dehydrant,  23. 
Acid,  hydrolysis  by,  i,  2,  8,  12,  36,  60. 

—  production  of,  in  invertase  action,  49. 
in  trypsin  action,  35,  48. 

Active  oxygen,  71,  72. 
Adsorption,  14,  59. 

—  affinity,  18,  62. 

—  compounds,  16,  59. 

—  law  of,  16,  59. 

—  of  charged  colloids,  16,  17,  63. 

—  of  enzymes,  19,  33,  62. 

—  specific,  61,  70. 
Alanine,  41. 

Alcohol  as  precipitant,  23. 

—  production  from  sugar,  7,  9. 
Alkali,  effect  on  rate  of  trypsin  action,  48. 
Amino-acids,  electrical  conductivity  of,  25. 

—  formed  by  pepsin,  9. 
trypsin,  9,  24,  26. 

—  protecting  trypsin  from  destruction,  n, 

43- 

—  retarding  action  of  trypsin,  35. 
Amylase,  6,  8,  n,  15,  19,  26,  29,  32,  42,  54, 

58,  74- 

—  not  protein  nor  carbohydrate,  19. 
Anti-enzymes,  33,  63,  66,  67. 

—  as  synthesizing  agents,  33. 
Antiseptics,  20,  58. 

—  action  on  enzymes,  20,  58. 

—  to  distinguish  enzymes  from  living  cells, 

20,  58. 

Anti-pepsin,  66,  68. 

Anti-toxins,  17,  66. 

Anti-trypsin  of  intestinal  worms,  66. 

•  —  of  serum  and  of  egg-white,  67. 

Arbutin,  44. 

Asparagine,  effect  on  amylase,  58. 

Assimilation  of  CO2  and  its  optimum  tem- 
perature, 52,  53. 

Autocatalysis,  in  ester  hydrolysis,  49. 

—  in  lactone  formation,  49. 

—  positive  and  negative,  49,  75. 
Autolytic  enzyme  in  "pepsin"  and  "tryp- 
sin,"  76. 

Autolysis  in  preparation  of  invertase,  21. 

BILE-SALTS  as  co-enzyme  for  lipase,  66. 
Biological  method  of  purifying  enzymes,  22. 
Bi-rotation  of  glucose,  26. 
Biuret  reaction,  25. 
Blood,  coagulation  of,  73. 

CALCIUM  salts,  effect  on  enzymes  and  zymo- 
gens,  63,65,  69,73. 


Carbohydrate  constituents  of  some  enzymes, 
19. 

Carbon  dioxide  assimilation,  52,  53,  73. 

Catalase,  71,  73. 

Catalysis,  distinguished  from  chemical  com- 
bination in  ratio  of  combining  pro- 
portion, 3. 

—  final   result   independent  of  concentra- 

tion of  catalyst,  3,  10. 

—  in  general,  i. 

—  negative,  2. 

Catalysts,  change  during  reaction,  3,  33. 

—  definite  chemical  bodies,  5,  20. 

—  definition  of,  i,  3. 

—  disappearance  during  reaction,  2,  n. 

—  minute  quantity  active,  3,12. 

—  non-appearance  in  final  products,  3. 
Catalytic  action  of  HC1  in  ester-formation,  4, 

5- 
Causes  of  acceleration,  46,  49. 

—  retardation,  35,  39,  46,  48,  49. 
Cell-activities  in  relation  to  enzymes,  20,  58. 
Charcoal,  occlusion  of  gases  by,  59. 
Chemical  changes  in  living  organisms,  i,  75. 

—  configuration  of  surfaces,  59,  61. 
Chlorophyll  function,  52,  73. 
Coagulation  of  blood,  73. 
Co-enzymes,  22,  57,  65,  69. 
Colloidal  characters  of  enzymes,  13,  20. 

—  complexes,  16. 

—  gold,  sizes  of  particles,  13. 

—  platinum,  3,  13,  52,  60. 
optimum  temperature,  52. 

—  solutions,  permanency  of,  17. 
Colloids,  action  of  electrolytes  on,  18. 

—  diffusibility  of,  14,  15,  18. 

—  electric  charge  of,  14,  15. 

—  general  properties  of,  13. 

—  hysteresis  of,  15. 

—  mutual  precipitation,  16,  18. 

—  osmotic  pressure  of,  14,  15,  18. 
Combination  between  catalyst  and  substrate, 

enzyme  and  substrate,  12,  33,  40,  42, 

44,  55,  59- 

products,  33,  40,  43,  49. 

Comparing  relative    strengths  of  enzyme- 
solutions,  24,  26,  55. 
Concentration  (evaporation)  of  solutions  of 

enzymes,  23. 
Concentration  of  enzyme,  effect  on  rate  of 

change,  42,  48,  55. 
as  affecting  final  result,  10. 

—  substrate,  as  affecting  rate  of  change,  42, 

49.  55- 
Conductivity,  electrical,  as  method,  25,  26. 


87 


88 


INDEX 


Configuration  of  surface  in  relation   to  ad- 
sorption, 61,  62. 

Congo-red,  14,  15,  17,  18,  60,  61,  63. 
Copper-reducing  power  as  method,  24. 
Coupled  reactions,  72. 

DESTRUCTION  of  enzyme  during  reaction,  n, 

33,  34.  39,  49,  54- 
Dextrin,  synthesis  of,  32,  55. 
Dextrinase,  15,  32,  54,  74. 
Diastase,  6,  15,  19,  54. 
Diffusibility  of  colloids,  14,  15,  18. 

—  enzymes,  13,  15,  20. 

Diffusion  factor  in  heterogeneous  reactions, 

5°- 

—  temperature  coefficient  of,  50. 
Dilatometer,  as  method,  25. 

Directive  action  of  catalyst  and  enzyme,  2, 

9- 

Disintegration    of    cells    for    extraction    of 
enzymes,  22. 

ELECTRIC  charge  of  colloids,  14,  15. 

enzymes,  15,  16,  62,  63. 

solids  in  water,  15,  17. 

Electrical  conductivity  of  amino-acids,  25. 

as  method,  25,  26. 

in  trypsin  action,  25. 

Electrolytes,  action  on  colloids,  18,  23. 

enzymes,  57,  63,  66. 

Electro-negative  colloids,  15. 
Electro-positive  colloids,  15. 
Emulsin,  12,  24,  31,  33,  40,  42,  44,  45,  50,  52, 
61,  74. 

—  in  yeast,  32. 
Enterokinase,  n,  69. 
Enzyme-action,  reversibility  of,  28,  75. 
Enzymes,  action  of  antiseptics,  20. 

—  as  catalysts,  6. 

—  as  colloids,  13,  19,  57,  59. 

—  as  properties  of  matter,  20. 

—  chemical  nature  of,  13,  19,  70. 

—  destroyed  by  boiling,  12,  20. 

—  diffusibility,  13,  20. 

—  electric  charge  of,  15,  16,  62,  63. 

—  instability  of,  n,  15,  19,  21,  33. 

—  intra-cellular,  21. 

—  name  suggested  by  Kuehne,  6. 

—  not  nucleo-proteins,  19. 

—  optical  activity  of,  19,  41,  70. 

—  preparation  of,  21. 

—  "  radio-activity  "  of,  20. 

—  specificity  of,  31,  40,  61,  72. 
Equation  for  enzyme  action,  50. 
Equilibrium,   alteration    of,    when    catalyst 

changes  during  reaction,  4,  33,  50,  61. 

—  as  affected  by  nature  or  concentration 

of  catalyst,  4,  12. 
—  definition  of,  4,  45,  47. 

—  unaffected  by  "  intensity- factor,"  50. 

—  Van't  Hoff  on,  31,  76. 

—  with  emulsin,  32. 

—  with  enzymes  in  general  a  genuine  one, 

33.  46- 

—  with  invertase,  12,  28,  46. 

—  with  lipase,  29,  30,  45,  48. 

—  with  maltase,  32,  55. 

—  with  trypsin,  33,  35. 
Erepsin,  8,  21,  34,  74. 


Ester-hydrolysis  in    heterogeneous    system, 

50,  60. 

Ethyl-butyrate,  synthesis  of,  12,  29. 
Evaporation  of  solutions  of  enzymes,  23. 
Exponential  law  of  adsorption,  16,  17,  59. 
enzyme-concentration  and  action,  56, 

60. 
Extinction  temperature,  54. 

FARADAY-TYNDALL  phenomenon,  13. 
Fat  absorption,  30. 

—  synthesis,  30. 

Ferments,  soluble  and  organised,  6. 
Fibrin,  19,  24,  70. 

—  an  adsorption-compound,  73. 
Fibrin-ferment,  73. 

Final    result    of   reaction    independent    of 

amount  of  enzyme,  n. 
Formaldehyde,  action  on  enzymes,  20. 

—  method  of  Sorensen,  23. 

—  produced  in  photo-synthesis,  73. 
Freezing-point  determinations,  as  method,  25. 

GELATIN,  action  of  trypsin  on,  34,  25,  55,  57. 
General  equation  for  enzyme-action,  50. 
Gentianose,  74. 

Glucosides,  stereochemistry  of,  30,  61. 
Glycogen  formation,  28,  29. 
Glycyl-tyrosine  hydrolysed  by  yeast-juice,  44. 

HAEMOGLOBIN  diffuses  into  gelatin,  15,  18. 
Heat,  action  on  enzymes,  12,  20,  26,  64. 
Heterogeneous  systems,  diffusion-factor,  50. 

reactions  in,  50,  60,  75. 

Homogeneous  systems,  reactions  in,  50. 

Hormones,  66. 

Hydrogen  peroxide,  3,  5,  7,  60,  71. 

in  photo-synthesis,  73. 

Hydrolysis  by  acids  contrasted  with  that  by 
enzymes,  8,  28,  29,  60,  76. 

—  in  heterogeneous  system,  50. 

—  of  cane-sugar,  8,  36,  38,  49,  60. 

—  caseinogen,  8. 

—  starch  by  water,  8. 
Hydrolyte,  7. 
Hysteresis  in  colloids,  15. 

INSTABILITY  of  enzymes,  u,  15,  19,  33. 
Integration,  37. 
Intensity-factor,  46,  48,  49,  51. 
Intermediate-compounds,  4,  12,  40,  41,  44. 

not  universal  explanation,  5. 

Intra-cellular  enzymes,  21. 

Investigation    of    enzyme-action,     general 

methods,  23. 
Invertase,  12,  15,  19,  26,  28,  40,  42,  46,  49, 

5°.  74- 

—  action  of  heat  on,  42. 

—  positive  auto-catalysis  in,  49. 

—  preparation  of,  21. 

—  reversibility  of,  12,  28. 
—  velocity  of  reaction,  38. 

Iron,  catalyst  for  oxidation,  i,  72. 
Isolactose,  32. 
Isomaltose,  31. 

LACCASE,  65,  72. 

Lactacidase,  74. 

Lactase,  velocity  of  reaction,  38,  42. 


INDEX 


89 


Lactic  acid,  oxidised  by  peroxide  and  per- 

oxidase,  72. 
Law  of  adsorption,  16. 
Leucine,  41. 

Leuco-base  of  malachite-green,  25. 
Linear  law  of  rate  of  change,  42. 

enzyme-concentration,  56. 

Lipase,  12,  25,  29,  30,  41,  46,  50,  60,  63,  65. 

—  reversibility  of,  12,  29,  30,  48. 

—  solubilities  of,  60,  63. 

Living    organism,    peculiarity    of   chemical 

changes  in,  I. 

"  Lock  and  key  "  simile,  40,  61. 
Logarithmic  curve,  37. 
-  law,  37,  46. 

MALACHITE-GREEN,  leuco-base  of,  25. 
Maltase,  12,  31,  32,  40,  42,  55,  61. 

—  synthesis  by,  12,  29,  31,  32. 
Maltose,  synthesis  of,  12,  31,  32. 
Mandelic  esters,  41. 
Manganese  and  laccase,  65. 

—  as  catalyst  in  oxidations,  i,  72. 
Mass-action,  law  of,  36. 

Mathematical  formulae,  value  of  in  biological 

science,  36,  51. 
Methyl-acetate,  28. 
Methyl-glucosides,  31,  44,  61. 
Mett's  tubes,  24,  50. 
Mixtures  of  enzymes,  32. 
Model  to  illustrate  catalysis,  2,  9. 
Myrosin,  25. 

NATURE  of  combination  between  enzyme  and 

substrate,  33,  41,  59,  75. 
Negative  auto-catalysis,  49. 
Negative  catalysis,  2. 
Nicotine  as  optically  active  catalyst,  77. 
Night-blue,  15,  17. 
Non-antagonism  of  physical    and  chemical 

points  of  view,  18. 
Nucleo-proteins,  19. 

OPTICAL  activity  as  method,  24. 

of  enzymes,  19,  41,  70. 

of  organic  catalysts,  77. 

Optical  factor,  24,  31. 

Optimum  temperature,  12,  52,  54,  75. 

in  case  of  colloidal  platinum,  52. 

Osmotic  pressure  of  colloids,  14,  15,  18. 
Oxidation  systems,  71. 
Oxydase,  72. 

PARANUCLEIN,  synthesis  of,  34. 

Pectase,  65. 

Pepsin,  8,  19,  20,  21,  25,  34,  57,  69. 

Pepsinogen,  69,  70. 

Peptones,  electrical  conductivity  of,  25. 

—  production  of  in  enzyme  actions,  9,  26. 
Permanency  of  colloidal  solutions,  17. 
Permolybdic    acid    formed  as   intermediate 

compound,  5. 
Peroxidase,  25,  71,  72. 
Peroxides,  71. 

Phosphates  as  activators  of  yeast-juice,  66. 
Phosphorus,  separation  from  caseinogen  by 

trypsin  and  by  alkali,  9. 

—  absent  from  pepsin,  19. 


Physical  consistency,    changes  in,  not  best 

method,  24. 
Plastein,  34. 

Polarimeter,  as  method,  24,  27. 
Polypeptides,  action  of  trypsin,  etc.,  on,  8, 35, 

41,  76. 

—  as  substrate,  24,  35,  41. 
Positive  auto-catalysis,  49. 
Precipitation  of  enzymes,  23. 
Preparation  of  enzymes,  21. 

Products,  retardation  of  reaction  by,  35,  40. 

Proportionality  of  final  result  to  concentra- 
tion of  enzyme,  n. 

Protamine,  synthesis  of,  34. 

Protection  of  enzyme  from  heat-destruction 
by  substrate  or  products,  42,  43. 

Protection  of  alimentary  canal  from  action 
of  enzymes,  68. 

Protein  reactions  of  certain  enzymes,  19. 

Proteoses,  production  by  enzymes,  26. 

Prothrombin,  73. 

Protoplasmic  activity  and  antiseptics,  20,  58. 

"RADIO-ACTIVITY  "  of  enzymes,  20. 

Raffinose,  74. 

Refractive  index  as  method,  25. 

Rennet,  12,  34,  63. 

Resistance  of  mucous  membrane  to  en- 
zymes, 68. 

Result  of  enzyme-action  independent  of 
amount  of  catalyst,  n. 

Retardation  by  products  of  reaction,  35,  40. 

—  causes  of,  35,  40. 
Reversible  reactions,  4,  45. 

as  catalysed  by  enzymes,  12. 

effect  of  catalysts  on,  4. 

Reversibility  in  relation  to  action  of  pro- 
ducts, 35,  43. 

—  of  amylase,  29,  32. 

—  of  emulsin,  12,  32. 

—  of  invertase,  12,  28. 

—  of  lipase,  12,  29. 

—  of  maltase,  12,  29,  31. 
Revertose,  31,  32. 

SALICIN,  synthesis  of,  12,  45. 

Side-chain  theory,  63. 

Sinigrin,  25. 

Slow  combination  of  oxygen-hydrogen  gas,  3. 

Solid  solution,  59. 

Solubilities  of  enzymes,  19,  60,  63. 

Specificity  of  adsorption,  61,  70. 

—  of  enzymes,  31,  40,  61,  72. 
Spectro-photometer  as  method,  25. 
Square-root  law  of  Schiitz,  55. 
Stability  of  enzymes,  n,  15,  19,  21,  33. 
Stages  of  reactions,  26. 

Starch,  action  of  acids  on,  8. 
diastase  on,  8,  42,  55. 

—  formation,  29. 

Stereo-chemistry  of  glucosides,  30. 
Stopping  a  reaction,  methods  of,  26. 
Substrate  defined,  7. 
Supersaturated  solutfons,  2. 
Surface  condensation,  5,  59. 

—  in  colloidal  solution,  14. 
Surface-tension,  14. 
Synthesis  by  lipase,  12,  29,  30. 

—  importance  of  small  amount,  29. 


9o 


INDEX 


Synthesis  of  dextrin,  32. 

-  of  ethyl-butyrate,  12,  29. 

—  of  higher  fats,  30. 

—  of  saccharose,  28,  46. 

—  of  salicin,  12,  45. 

—  of  starch,  28,  29. 

Synthetic  action  of  proteoclastic  enzymes,  34. 

—  enzymes,  33. 

TEMPERATURE-COEFFICIENT  of  diffusion,  50. 

of  a  physical  process,  50,  61. 

Temperature,  effect  on  ionisation  of  water,  8. 

—  effect  on  velocity  of  enzyme-action,  26, 

52,  61. 

—  of  destruction  of  enzymes,  12,  20,  54. 

—  optimum,  12,  52,  54,  75. 

-  Van't  Hoff  s  rule  of  effect  of,  52,  53. 
Terminology  of  enzymes,  7. 
Thrombase,  73. 

Thrombogen,  73. 

Thrombokinase,  73. 

Time-factor  in  destruction  of  enzymes,  etc., 

52. 

Toluene  as  antiseptic,  20,  58. 
Toxoids,  63. 
Trigger  action,  2,  9. 
Trypsin,  8,  10,  15,  19,  21,  23,  24,  25,  26,  34, 

35,  38,  41,  48,  52,  55,  56,  57,  58,  59, 

63,  64,  76. 

—  effect  of  alkali  on  rate  of  action,  48. 


Trypsin  negative  auto-catalysis,  48. 
-    not  activator  of  OH  ions,  9. 
—  velocity  of  reaction,  10. 
Trypsinogen,  n,  21,  69. 
Tyrosinase,  72. 
Tyrosine,  41. 

ULTRA-MICROSCOPE  in  enzyme-action,  15,  62. 
Unimolecular  reaction,  formula  for,  37. 
Uranyl-phosphate   method   of  precipitation, 

23,  70- 
Uricolytic  enzyme,  22. 

VALENCY  in  effect  of  electrolytes  on  colloids, 

18. 

Velocity-constant  of  reactions,  37. 
Velocity  of  reaction,  36. 
Viscosity,  as  method,  24,  27. 

—  of  proteins,  24. 

YEAST,  emulsin  in,  32. 

—  preparation  of  enzymes  from,  21. 

—  press-juice,  action  on  glycyl-tyrosine,  44. 
co-enzyme  of,  65. 

ZYMASE,  n,  22,  23,  58,  74,  76. 

—  co-enzyme  of,  65. 

—  preparation  of,  21,  22. 
Zymogens,  n,  21,  69. 
Zymoids,  63. 


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